<p class="jove_content">This study was supported by the National Institutes of Health (NIH) of the United States (RO1 AI68002).</p>

<p class="jove_title">1.  Recombinant SARS-CoV RBD Protein Preparation</p>
<ol>
<li>Prepare calcium phosphate transfection reagent
<ol>
<li>2X HBS buffer preparation: Mix together 16 g of NaCl, 0.4 g of Na<sub>2</sub>HPO<sub>4</sub>*7H<sub>2</sub>O, and 13.0 g of HEPES. Adjust pH to 7.00 and bring up total volume to 1000  mL in distilled water. After filtering the solution for sterilization, aliquot and store it at -20&deg;C.<br />
Hint: Any variation of the pH value would affect the transfection results. Thus, it is advisable to test several pH values around 7.00 (for example, 6.99, 7.00, or 7.01) and find the best one for the transfection using the method introduced below.</li>
<li>2.5 M CaCl<sub>2</sub> preparation: Add 73.5 g of CaCl<sub>2</sub>*2H<sub>2</sub>O to distilled water in a final volume of 200  mL. Filter the solution and store at -20&deg;C.</li>
</ol>
</li>
<li>Recombinant plasmid transfection and protein purification
<ol>
<li>Split 293T cells at 50-70% confluency 24 h before transfection. Grow cells in T-175 cm<sup>2</sup> tissue culture flasks in 40 mL DMEM containing 10% heat-inactivated (HI) FBS and 1% Penicillin/Streptomycin (P/S) at 37&deg;C in 5% CO<sub>2</sub>.</li>
<li>All transfection reagents should be brought up to room temperature before transfection. These reagents include 2X HBS, 2.5M CaCl<sub>2</sub>, DiH<sub>2</sub>O, and rRBD plasmid <sup>6</sup>.<br />
Hint: The final recombinant plasmid construct used for protein expression should contain a signal peptide to ensure secretion of expressed recombinant proteins to the culture supernatant. A 6x His tag may be added to the C-terminal of the expressed protein for easy purification.</li>
<li>Prepare one 50  mL (A) and one 15  mL (B) BD Falcon tube, and add reagents to the tubes as indicated in Table 1. Add 2X HBS buffer to tube A. In tube B, add 2.5M CaCl<sub>2</sub> and rRBD plasmid, and bring the volume to the requirement in DiH<sub>2</sub>O.<br />
  <table border="1">
<tbody>
    <tr>
      <td colspan="4"><strong>A. One T-175 cm<sup>2</sup> tissue   culture flask (4000  &mu;L/flask): prepare for rRBD expression</strong></td>
    </tr>
    <tr>
      <td colspan="2"><strong>Tube A </strong></td>
      <td colspan="2"><strong>Tube B</strong></td>
    </tr>
    <tr>
      <td>2X HBS</td>
      <td>2000  &mu;L</td>
      <td>2.5M   CaCl<sub>2</sub></td>
      <td>200  &mu;L</td>
    </tr>
    <tr>
      <td>&nbsp;</td>
      <td>&nbsp;</td>
      <td>rRBD   plasmid DNA</td>
      <td>40 &mu;g</td>
    </tr>
    <tr>
      <td>&nbsp;</td>
      <td>&nbsp;</td>
      <td><em>DiH<sub>2</sub>O to</em></td>
      <td><em>2000  &mu;L</em></td>
    </tr>
    <tr>
      <td colspan="4"><strong>B. One 100-mm petri dish (1000    &mu;L/dish): prepare for SARS pseudovirus production</strong></td>
    </tr>
    <tr>
      <td colspan="2"><strong>Tube A </strong></td>
      <td colspan="2"><strong>Tube B</strong></td>
    </tr>
    <tr>
      <td>2X HBS</td>
      <td>500  &mu;L</td>
      <td>2.5M   CaCl<sub>2</sub></td>
      <td>50  &mu;L</td>
    </tr>
    <tr>
      <td>&nbsp;</td>
      <td>&nbsp;</td>
      <td>SARS-CoV   S plasmid DNA</td>
      <td>5 &mu;g</td>
    </tr>
    <tr>
      <td>&nbsp;</td>
      <td>&nbsp;</td>
      <td>HIV-1 plasmid (pNL4-3.luc.RE)</td>
      <td>5 &mu;g</td>
    </tr>
    <tr>
      <td>&nbsp;</td>
      <td>&nbsp;</td>
      <td><em>DiH<sub>2</sub>O to</em></td>
      <td><em>500  &mu;L</em></td>
    </tr>
  </tbody>
</table>
<strong>Table 1.</strong> Transfection mixture preparation and volumes<br />
The volumes listed in Table 1 are for one transfection unit. If more flasks or dishes are used for the transfection, adjust volumes accordingly.</li>
<li>Add the DNA-calcium solution in tube B into tube A in a dropwise manner, while maintaining constant and gentle mixing in a vortex. Let the mixture sit at room temperature for 20-30 min. The key for successful calcium phosphate transfection depends on the size and shape of the precipitate formed. Thus, the mixture should be vortexed constantly and slowly to meet this requirement and, as a result, improve transfection efficacy.</li>
<li>Add mixture in a dropwise and even manner into 293T cells (4000  &mu;L/flask). Culture cells in incubator at 37&deg;C in 5% CO<sub>2</sub>.</li>
<li>Replace the culture medium with fresh serum-free OPTI-MEM I Reduced-Serum Medium (50  mL/flask) 8-10 h post-transfection. Continue to culture cells for two more days in the same condition.</li>
<li>Collect supernatant containing expressed rRBD protein 72 h post-transfection. Centrifuge at 6000 rpm for 15 min to remove cell debris. Add protease inhibitor cocktail to the collected supernatant and store at 4&deg;C overnight.</li>
<li>On the next day, purify rRBD recombinant protein from the culture supernatant using Ni-NTA Superflow following manufacturer's instructions.</li>
<li>Concentrate purified protein using Amicon Ultra -15 concentration tubes. After protein concentration, add PBS to the concentration tubes and centrifuge again to remove imidazole in elution buffer. Calculate protein concentration and store purified protein at -80&deg;C until use.</li>
</ol>
</li>
</ol>
<p class="jove_title">2.  Mouse Immunization and Sample Collection</p>
<ol>
<li>Pre-warm Sigma adjuvant system (SAS) to 40-45&deg;C according to manufacturer's instructions. Add 1  mL PBS per vial and mix thoroughly.</li>
<li>Prepare protein-adjuvant emulsion according to the protocol in Table 2. Pipette calculated rRBD protein into a 1.5  mL tube. Add equal volume of SAS adjuvant to the tube and vortex vigorously for 2-3 min to form emulsion.<br />
Hint: The volumes listed in Table 2 are for one mouse. Adjust volumes according to the actual mouse numbers used. For each group, mix together the proteins and adjuvant required for each vaccine. Always prepare one extra sample for the vaccination to ensure accuracy.<br />
<table border="1">
<tbody>
  <tr>
    <td><strong>Group</strong></td>
    <td><strong>1<sup>st</sup> vaccine </strong><br />
      (200    &mu;L/mouse)</td>
    <td><strong>2<sup>nd</sup> vaccine </strong><br />
      (200    &mu;L/mouse)</td>
    <td><strong>3<sup>rd</sup> vaccine </strong><br />
      (200    &mu;L/mouse)</td>
  </tr>
  <tr>
    <td>rRBD   protein</td>
    <td>20 &mu;g   protein in PBS <br />
      (100    &mu;L) + 100  &mu;L SAS</td>
    <td>10 &mu;g   protein in PBS (100  &mu;L) + 100  &mu;L SAS</td>
    <td>10 &mu;g   protein in PBS (100  &mu;L) + 100  &mu;L SAS</td>
  </tr>
  <tr>
    <td>PBS   control</td>
    <td>100  &mu;L   PBS + <br />
      100  &mu;L   SAS</td>
    <td>100  &mu;L   PBS + <br />
      100  &mu;L   SAS</td>
    <td>100  &mu;L   PBS + <br />
      100  &mu;L   SAS</td>
  </tr>
</tbody>
</table>
<strong>Table 2.</strong> Mouse immunization protocol</li>
<li>Subcutaneously prime-immunize female BALB/c mice (4-6 weeks old, 5 mice/group), and boost twice with rRBD and SAS as indicated in Table 2. Use PBS group as the control. The site for subcutaneous injections usually chosen is the loose skin between the shoulder blades. Alternatively, the ventral abdomen is commonly used, because it is easier to inject, and may observe any leakage from the injection sites. When repeated doses of vaccines are used, it is easy to select various injection sites, preventing potential local skin reactions.</li>
<li>Bleed mice via retro-orbital with anesthetic before immunization and 10 days after each vaccination, and heat sera at 56&deg;C for 30 min to inactivate complement. Store mouse sera at -20&deg;C until use.</li>
</ol>
<p class="jove_title">3.  Neutralization Detection using Pseudovirus Neutralization Assay</p>
<ol>
<li>Prepare SARS pseudovirus
<ol>
<li>Split 293T cells in 100 mm tissue culture petri dishes (2x10<sup>6</sup> cells/dish) 16 h before transfection, and grow cells as above.</li>
<li>Prepare transfection reagents as indicated in Table 1. Co-transfect a plasmid encoding SARS-CoV S protein and a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) using calcium phosphate transfection reagent.</li>
<li>Replace medium with 10  mL fresh DMEM containing 10% FBS and 1% P/S 8-10 h post-transfection. Collect supernatant containing SARS pseudovirus 72 h post-transfection.</li>
<li>Filter pseudovirus through a 0.45 &mu;m filter. Aliquot and store at -80&deg;C until use.</li>
</ol>
</li>
<li>SARS pseudovirus neutralization assay
<ol>
<li>Split 293T cells expressing SARS-CoV receptor ACE2 (ACE2/293T) at 10<sup>4</sup> cells/100  &mu;L/well in 96-well tissue culture plates 16 h before infection.</li>
<li>Dilute SARS pseudovirus by 2-fold to detect the virus titer in ACE2/293T cells.</li>
<li>Serially dilute mouse sera in 96-well tissue culture plates, and add equal volume of titrated SARS pseudovirus. Preincubate the plates at 37&deg;C for 1 h. </li>
<li>After incubation, add 100  &mu;L of the sera-pseudovirus mixture to ACE2/293T cells, and continue to grow cells at 37&deg;C in 5% CO<sub>2</sub>. Add fresh DMEM 24 h later.</li>
<li>Completely remove culture supernatants from the plates 72 h post-infection. Add 1X luciferase cell culture lysis reagent (60  &mu;L/well), and promote cell lysis with constant shaking of plates for 1-2 h at room temperature.</li>
<li>Transfer cell lysates (50  &mu;L/well) into luminometer plates (Microfluor 96-well plates). Add luciferase substrate (50  &mu;L/well) included in luciferase assay system, and detect relative luciferase activity in Ultra 384 luminometer.</li>
<li>Calculate SARS pseudovirus neutralization titer and present as 50% neutralizing antibody titer (NT<sub>50</sub>) <sup>6</sup>.</li>
</ol>
</li>
</ol>

<ol><li>Pitcovski, J., Gutter, B., Gallili, G., Goldway, M., Perelman, B., Gross, G., Krispel, S., Barbakov, M., Michael, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+large-scale+use+of+recombinant+VP2+vaccine+for+the+prevention+of+infectious+bursal+disease+of+chickens%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens.</a> <em>Vaccine</em>. <strong>21</strong>, 4736-4743 (2003).</li>
<li>Tait, A., Hall, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Theileria+annulata%3A+control+measures%2C+diagnosis+and+the+potential+use+of+subunit+vaccines%5BTitle%5D)+AND+%22Rev.+Sci.+Tech%22%5BJournal%5D)">Theileria annulata: control measures, diagnosis and the potential use of subunit vaccines.</a> <em>Rev. Sci. Tech</em>. <strong>9</strong>, 387-403 (1990).</li>
<li>Qi, Z., Zhou, L., Zhang, Q., Ren, L., Dai, R., Wu, B., Wang, T., Zhu, Z., Yang, Y., Cui, B., Wang, Z., Wang, H., Qiu, Y., Guo, Z., Yang, R., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+mouse%2C+guinea+pig+and+rabbit+models+for+evaluation+of+plague+subunit+vaccine+F1%2BrV270%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">Comparison of mouse, guinea pig and rabbit models for evaluation of plague subunit vaccine F1+rV270.</a> <em>Vaccine</em>. <strong>28</strong>, 1655-1660 (2010).</li>
<li>Rosser, M. P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P., Cobb, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+transfection+of+CHO-K1-S+using+serum-free+medium+in+suspension%3A+a+rapid+mammalian+protein+expression+system%5BTitle%5D)+AND+%22Protein+Expr.+Purif%22%5BJournal%5D)">Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system.</a> <em>Protein Expr. Purif</em>. <strong>40</strong>, 237-243 (2005).</li>
<li>Du, L., Zhao, G., Li, L., He, Y., Zhou, Y., Zheng, B. J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigenicity+and+immunogenicity+of+SARS-CoV+S+protein+receptor-binding+domain+stably+expressed+in+CHO+cells%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells.</a> <em>Biochem. Biophys. Res. Commun</em>. <strong>384</strong>, 486-490 (2009).</li>
<li>Du, L., Zhao, G., Chan, C. C., Sun, S., Chen, M., Liu, Z., Guo, H., He, Y., Zhou, Y., Zheng, B. J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombinant+receptor-binding+domain+of+SARS-CoV+spike+protein+expressed+in+mammalian%2C+insect+and+E.+coli+cells+elicits+potent+neutralizing+antibody+and+protective+immunity%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.</a> <em>Virology</em>. <strong>393</strong>, 144-150 (2009).</li>
<li>Ambrosino, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amino+acids+270+to+510+of+the+severe+acute+respiratory+syndrome+coronavirus+spike+protein+are+required+for+interaction+with+receptor%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor.</a> <em>J. Virol</em>. <strong>78</strong>, 4552-4560 (2004).</li>
<li>Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., Choe, H., Farzan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiotensin-converting+enzyme+2+is+a+functional+receptor+for+the+SARS+coronavirus%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.</a> <em>Nature</em>. <strong>426</strong>, 450-454 (2003).</li>
<li>He, Y., Zhou, Y., Liu, S., Kou, Z., Li, W., Farzan, M., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Receptor-binding+domain+of+SARS-CoV+spike+protein+induces+highly+potent+neutralizing+antibodies%3A+implication+for+developing+subunit+vaccine%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine.</a> <em>Biochem. Biophys. Res. Commun</em>. <strong>324</strong>, 773-781 (2004).</li>
<li>Batard, P., Jordan, M., Wurm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfer+of+high+copy+number+plasmid+into+mammalian+cells+by+calcium+phosphate+transfection%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection.</a> <em>Gene</em>. <strong>270</strong>, 61-68 (2001).</li>
<li>Pramfalk, C., Lanner, J., Andersson, M., Danielsson, E., Kaiser, C., Renstrom, I. M., Warolen, M., James, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+receptor+activation+and+down-regulation+by+cationic+lipid+transfection+reagents%5BTitle%5D)+AND+%22BMC.+Cell+Biol%22%5BJournal%5D)">Insulin receptor activation and down-regulation by cationic lipid transfection reagents.</a> <em>BMC. Cell Biol</em>. <strong>5</strong>, 7-7 (2004).</li>
<li>Rosser, M. P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P., Cobb, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+transfection+of+CHO-K1-S+using+serum-free+medium+in+suspension%3A+a+rapid+mammalian+protein+expression+system%5BTitle%5D)+AND+%22Protein+Expr.+Purif%22%5BJournal%5D)">Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system.</a> <em>Protein Expr. Purif</em>. <strong>40</strong>, 237-243 (2005).</li>
<li>Jiang, M., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+Ca2%2B-phosphate+transfection+efficiency+in+low-density+neuronal+cultures%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.</a> <em>Nat. Protoc</em>. <strong>1</strong>, 695-700 (2006).</li>
<li>Jiang, M., Deng, L., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+Ca%282%2B%29-phosphate+transfection+efficiency+enables+single+neuron+gene+analysis%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">High Ca(2+)-phosphate transfection efficiency enables single neuron gene analysis.</a> <em>Gene Ther</em>. <strong>11</strong>, 1303-1311 (2004).</li>
<li>Han, D. P., Kim, H. G., Kim, Y. B., Poon, L. L., Cho, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+safe+neutralization+assay+for+SARS-CoV+and+characterization+of+S-glycoprotein%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein.</a> <em>Virology</em>. <strong>326</strong>, 140-149 (2004).</li>
<li>Toledo, J. R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors%5BTitle%5D)+AND+%22Appl.+Biochem.+Biotechnol%22%5BJournal%5D)">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> <em>Appl. Biochem. Biotechnol</em>. <strong>157</strong>, 538-544 (2009).</li>
</ol>

<p class="jove_content">No conflicts of interest declared.</p>

<p class="jove_content">Cell density is an important factor affecting the efficacy of calcium phosphate-based transfection. In our experience, less than 70% confluency of the cells brings the best results. Thus, in order to improve transfection efficiency, it is recommended that cell density be kept at around 50-70% of confluency. The calcium phosphate transfection method is generally thought to be less efficient compared with other transfection methods, such as lipid transfection <sup>16</sup>. However, in this protocol, we used a modified calcium phosphate transfection method whereby constant and slow mixing of the transfection solution in a vortex ensures the formation of a precipitate with the appropriate size and shape, thus improving transfection efficiency.</p>

<table><tbody><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S2429</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C5080</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>12430</td><td></td></tr><tr><td>HI FBS</td><td>Invitrogen</td><td>10438</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140</td><td></td></tr><tr><td>OPTI-MEM I Medium</td><td>Invitrogen</td><td>31985</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche Group</td><td>11836170001</td><td></td></tr><tr><td>Ni-NTA Superflow</td><td>Qiagen</td><td>30450</td><td></td></tr><tr><td>Sigma adjuvant system</td><td>Sigma-Aldrich</td><td>S6322</td><td></td></tr><tr><td>Luciferase assay system</td><td>Promega Corp.</td><td>E1501</td><td></td></tr><tr><td>Luciferase cell culture lysis reagent (5X)</td><td>Promega Corp.</td><td>E1531</td><td></td></tr><tr><td>Amicon Ultra - 15</td><td>EMD Millipore</td><td>UFC901024</td><td></td></tr><tr><td>Microfluor 96-well plates</td><td>Thermo Fisher Scientific, Inc.</td><td>7905</td><td></td></tr><tr><td>Ultra 384 luminometer</td><td>Tecan Group Ltd.</td><td></td><td></td></tr></tbody></table>

protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection

<p class="jove_content">Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens <sup>1-3</sup>. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity <sup>4-6</sup>.</p>
<p class="jove_content">Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection <sup>7-9</sup>. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity <sup>6</sup>. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method <sup>6,10</sup> with some modifications. Compared with the lipid transfection method <sup>11,12</sup>, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed <sup>13,14</sup>. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory <sup>15</sup>.</p>
<p class="jove_content">The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.</p>

Watch this Scientific Journal Video about Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection at JoVE.com

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

<p class="jove_content">This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

</p>

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

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Immunology and Infection

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Video: Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Bacterial Transformation

<h4>Background</h4>

<p>In early 20<sup>th</sup> century, pneumonia was accountable for a large portion of infectious disease deaths<sup>1</sup>. In order to develop an effective vaccine against pneumonia, Frederick Griffith set out to study two different strains of the <em>Streptococcus pneumoniae: </em>a non-virulent strain with a rough appearance (R-strain) and a virulent strain with a smooth appearance (S-strain) due to an outer polysaccharide capsule<sup>2</sup>. This outer layer of the S-strain bacteria enabled them to withstand the host immune system, eventually leading to a life-threatening illness. When Griffith separately injected mice with heat-killed bacteria of either strain, the mice lived. However, when he injected mice with a combination of the heat-killed S-strain with live R-strain, the mice died<sup>2</sup>. When he analyzed the samples obtained from dead mice injected with the combination, he observed live S-strain bacteria to be present. In 1928, Griffith noted that a &#8220;transformation&#8221; process must have occurred to change the nonvirulent bacteria into the virulent strain. As the first known discovery of bacterial transformation, his discovery paved the way for the development of an essential tool in genetic engineering - transformation<sup>3</sup>.</p>

<p>Transformation is the genetic change in a cell due to the intake of DNA from the environment. In Griffith&#8217;s experiment, the DNA that encodes the protective polysaccharide coating of the S-strain bacteria did not break down from the heat shock and was introduced into the R-strain, allowing the latter to bypass the immune system of the mouse. While this process constantly occurs in the wild between organisms and even different species, scientists transform bacteria in laboratory settings for research purposes<sup>4</sup>.</p>

<h4>Bacterial Transformation using Plasmids</h4>

<p>Bacteria are the ideal organisms for transformation as they can easily take in exogenous genetic material into their genome and quickly amplify it<sup>3,5</sup>. They have one circular chromosome and multiple small circular pieces of double-stranded DNA called plasmids within the cytoplasm. These plasmids can replicate independently from the chromosomal DNA  and generally provide certain functional benefits, such as resistance to antibiotics<sup>6,7</sup>. In their natural environment, bacteria undergo &#8220;bacterial transformation&#8221; by taking in plasmids from other bacteria in a process called conjugation<sup>8</sup>. Moreover, when they multiply, each of their progeny receives a copy of the new plasmid.</p>

<p>Plasmids that are used for experimental purposes are called plasmid vectors. In a laboratory setting, scientists can artificially create &#8220;recombinant plasmids&#8221; that are approximately 5,000-10,000 base pairs in length by inserting DNA fragments into a plasmid vector. These recombinant plasmids usually have certain components: an origin of replication (ORI), an antibiotic resistance gene, a multiple cloning site, a promoter, a selection marker, and the gene of interest. The origin of replication is where replication begins. The antibiotic resistance gene allows the bacteria that take in the plasmid to survive on plates in the presence of a certain antibiotic drug. Although plasmids are relatively small pieces of DNA, scientists need to treat the host cells to enable the plasmid penetration through the cell membrane. Hence, the efficiency of the transformation is directly related to porosity of the host membrane.  One common approach is to heat-shock the bacteria that have been treated with a calcium chloride solution<sup>9</sup>. The bacteria that do not incorporate the plasmid will not transform, and therefore have no resistance to survive on the plate and be visible. The multiple cloning sites aid in DNA insertion by containing sites for restriction enzymes to cut the plasmid where the gene of interest can be inserted and ligated. The promoter drives transcription of the gene of interest. It is tagged by a marker, usually a fluorescent protein such as a green fluorescent protein (GFP), or can be an additional antibiotic resistance gene. The antibiotic resistance gene and the other selection markers help us determine whether the collected bacteria contain the plasmid of interest.</p>

<h4>Applications</h4>

<p>Effective transformation methods enabled scientists to isolate and profile genes and gene products and led to many advancements in life sciences and medicine, such as development of effective drugs, generation of genetically-modified crops, and advanced diagnostic tools<sup>10</sup>.  In addition, with technological advancements, new methods of transformation have emerged. For instance, Gateway Cloning allows insertion of multiple DNA fragments into different vectors as well as transfer of DNA sequences between plasmids<sup>11</sup>. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a gene-editing technique that directly modifies nucleotides in the genome and does not require the use of plasmids<sup>12</sup>. After transformation, researchers often isolate and profile the gene of interest and its products. Subsequently, the whole process of genetic cloning has opened up a new field of genetic manipulation. Thanks to genetic cloning, researchers can manipulate bacteria to produce large amounts of specific human proteins, such as insulin to treat diabetes patients<sup>10</sup>. Cloning also has served a major importance in modern day agriculture. Genetically modified organisms (GMO) are a direct result of genetic cloning and bacterial transformation<sup>10</sup>. For instance, scientists are working to generate genetically modified crops with nitrogen-fixing genes incorporated into their genome to boost food production and reduce fertilizer use, thus, diminishing the economic and environmental impact of fertilizers<sup>13</sup>. In sum, bacterial transformation is the first step of modern-day biotechnology and the foundation of future research discoveries.</p>

<h4>References</h4>

<ol class="references">
	<li><strong>GL, Armstrong, LA, Conn and RW, Pinner.</strong> Trends in Infectious Disease Mortality in the United States During the 20th Century. <em>JAMA. </em>1999, Vol. 281, 1 (61-66).</li>
	<li><strong>F, Griffith.</strong> The Significance of Pneumococcal Types. <em>J Hyg (Lond). . </em>1928 , 2 (113-59).</li>
	<li><strong>Lorenz, MG and Wackernagel, F.</strong> Bacterial gene transfer by natural genetic transformation in the environment. <em>Microbiol Rev. . </em>1994 Sep;, Vol. 58, 3: (563-602).</li>
	<li><strong>S, Domingues, et al.</strong> Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species. <em>PLOS Pathogens. </em>2012, 8(8): e1002837.</li>
	<li><strong>Dubnau, D.</strong> DNA uptake in bacteria. <em>Annu Rev Microbiol. . </em>1999, 53: (217-44).</li>
	<li><strong>Solar, G del, et al.</strong> Replication and control of circular bacterial plasmids. <em>Microbiol Mol Biol Rev. . </em>1998 , Vol. 62, 2: (434-64).</li>
	<li><strong>Bennett, PM.</strong> Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. <em>Br J Pharmacol. . </em>2008 , Vol. 153, S1: (S347-S357).</li>
	<li><strong>MLlosa, et al.</strong> Bacterial conjugation: a two-step mechanism for DNA transport. <em>Mol Microbiol. </em>2002 Jul;, Vol. 45, 1: (1-8).</li>
	<li><strong>Roychoudhury, A, Basu, S and Sengupta, DN.</strong> Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. <em>Indian J Biochem Biophys. . </em>2009 , Vol. 46, 5: (395-400).</li>
	<li><strong>Khan, S, et al.</strong> Role of Recombinant DNA Technology to Improve Life. I. <em>nt J Genomics. . </em>2016, 2016:2405954.</li>
	<li><strong>Marsischky, G and LaBaer, J.</strong> Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods. <em>Genome Res. </em>2004, Vol. 14, 2020-28.</li>
	<li><strong>Doudna, JA and Charpentie, E.</strong> The new frontier of genome engineering with CRISPR-Cas9. <em>Science. </em>2014, Vol. 346, 6213-1258096.</li>
	<li><strong>Good, A.</strong> Toward nitrogen-fixing plants. <em>Science. </em>2018, Vol. 359, 6378: 869-70.</li>
</ol>


Bacterial Transformation using Plasmids - Concept

Transformacja bakteryjna

Background

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine ...

Education

JoVE Lab Manual

Biology

Genetics

Buffers

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">To set up for this lab experiment, wear the appropriate personal protective equipment, including a lab coat, chemical splash goggles, and gloves. All solutions for this lab should be prepared in a chemical fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 100 mL of a 1 M NaOH solution. <strong>Note: </strong>NaOH is strongly corrosive, so use caution while handling it.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 4 g of solid NaOH and pour it in a 125-mL Erlenmeyer flask. Then, add 50 mL of deionized water to the flask. Stir the mixture on a stir plate to dissolve the NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label a 100-mL polyethylene bottle as &#39;1 M NaOH&#39;. Once the NaOH has dissolved and the solution appears homogeneous, add another 50 mL of deionized water. Then, retrieve the stir bar and transfer the solution to the labeled bottle. Place the capped bottle in the back of the instructor&#39;s hood.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Prepare 10 mL of a 0.56 mM solution of neutral red in deionized water. Measure 1.6172 mg of neutral red into a 10-mL volumetric flask.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use a pipette to fill the flask about 2/3 full with deionized water. Cover the flask with plastic paraffin film and agitate the mixture until the neutral red dissolves. Then, remove the film and fill the flask to the marked line. Place a micro-stir bar in the flask and stir the solution until it appears homogeneous.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Label a 20-mL vial as &#39;0.56 mM neutral red&#39; and transfer the solution to the vial. Cap the vial and store it in the fridge.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare 10 mL of a 0.66 mM solution of riboflavin-binding protein in the same way, starting by weighing 198 mg of the protein. Transfer the solution to a labeled 20-mL vial and store the solution in the fridge.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Prepare or obtain a set of buffers. Here, we will go through the preparation of 100 mL of 50 mM pH 6.5 monosodium phosphate buffer, adjusted with 1 M NaOH as an example.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure 0.5995 g of anhydrous monosodium phosphate into a 125-mL Erlenmeyer flask. Add 80 mL of deionized water to the flask and begin stirring the mixture.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">While the mixture stirs, turn on and calibrate a digital pH meter.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once the salt has dissolved and the solution appears homogeneous, clamp the pH sensor in the buffer and wait for the reading to stabilize.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Confirm that the 1 M NaOH solution has cooled to room temperature. Then, slowly add NaOH to the stirring buffer solution with pauses to let the pH stabilize until the buffer reaches pH 6.5. This may only take 1 or 2 mL of NaOH. Then, turn off the stir motor, rinse the pH probe, and retrieve the stir bar.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Pour the pH adjusted buffer into a clean 100-mL volumetric flask and bring the solution up to the 100-mL mark with deionized water. Seal the flask with plastic paraffin film and invert it until the solution appears homogeneous.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Label a clean 125-mL polyethylene bottle as &#39;50 mM monosodium phosphate buffer pH 6.5&#39;. Transfer the solution to the bottle and cap it. Store the buffer in the instructor&#39;s hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Prepare additional buffers in a similar fashion, using 1 M NaOH or 2 M HCl as appropriate to adjust the pH. Alter the buffer concentrations as needed to achieve roughly the same ionic strength.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">When finished, store the 1 M NaOH in a corrosives cabinet, clean the glassware and equipment using your standard procedures, and throw out disposable items in the lab trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Bring a container of anhydrous monosodium phosphate to the analytical balance area and confirm that there are sufficient supplies of weighing paper, laboratory wipes, and clean spatulas.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Set out the following lab equipment and glassware at each lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stir plate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab stand with thermometer clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;pH meter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Handheld spectrophotometer</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Calibration buffers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;400-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;100-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;25-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;10-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small watch glass</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Magnetic stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Forceps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Plastic wash bottle</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250- or 500-mL capped polyethylene bottle</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of laboratory tape</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Plastic pipettes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Labeling pen</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Box of laboratory wipes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;1-mL micropipette with tips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;200-&#181;L micropipette with tips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;10&#160;&#160;&#160;&#160;1.5-mL cuvettes with caps</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Set out additional disposable plastic pipettes so that everyone will have easy access to them.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Place labeled bottles or flasks containing the buffers in a central area.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label a 1-mL micropipette for each buffer and place the micropipettes with the corresponding buffers along with a box of pipette tips.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Portion out the neutral red and protein solutions. Label three 5-mL test tubes as &#39;neutral red&#39; and three 5-mL test tubes as &#39;riboflavin-binding protein&#39;. Then, take the vials of neutral red solution and riboflavin-binding protein from the fridge and place about 2 mL of the neutral red solution and 2 mL of riboflavin-binding protein solution in the appropriately labeled test tubes.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set out the neutral red and protein solutions so the student groups can share the test tubes.</div>
		</li>
	</ul>
	</li>
</ol>

Buffers: Common Ion Effect, Henderson-Hasselbalch Equation, and Buffer Capacity - Prep

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Preparation of SolutionsExpand
	Here, we show the laboratory preparation for 10 students ...

Chemistry

Lab Manual Sub Articles

Recrystallization

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Recrystallization of Acetanilide<button class="expand">Expand</button>
	<p>In this part of the lab, you&#39;ll purify acetanilide by recrystallization from an aqueous solution. Acetanilide is moderately soluble in boiling water, but it&#39;s much less soluble in room temperature and cold water. Aniline, a potential impurity, is much more soluble in room temperature water. Thus, you&#39;ll first make a saturated solution of acetanilide in boiling water. Then, you&#39;ll slowly cool the solution to room temperature.</p>
	<p>As the acetanilide&#39;s solubility decreases, it will slowly form flat colorless or white crystals. Water-soluble impurities will remain in solution. You will then cool the solution to further decrease the solubility of acetanilide. Finally, you&#39;ll recover the crystals with vacuum filtration and measure the melting point of your crystals.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before you start the lab, put on a lab coat, safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">To begin, bring a 125-mL Erlenmeyer flask and your lab notebook to the analytical balance to obtain your acetanilide.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Measure 2 g of acetanilide in a tared weighing boat and record the precise mass in your lab notebook. Add the acetanilide to the flask and bring it back to your fume hood.
		<h4><strong>Table 1: Recrystallization of acetanilide</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Compound</strong></td>
					<td><strong>Mass (g) </strong></td>
					<td><strong>Melting point (&#176;C) </strong></td>
				</tr>
				<tr>
					<td>Acetanilide</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Seed crystal (if used)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Recrystallized acetanilide<br />
					(<em>subtract seed crystal if used</em>)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td><strong>Percent recovery</strong></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/58/Table_1._Recrystallization_of_acetanilide.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Then, measure 30 mL of deionized water and carefully pour it into the flask of acetanilide.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Place the flask on a stirring hotplate, add a stir bar, and set the hotplate to medium heat and stirring. Let the mixture boil for 4 &#8211; 5 min to dissolve the acetanilide.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">If you see solid acetanilide after the solution has boiled for 5 min, measure another 10 mL of deionized water. Use a Pasteur pipette to add water dropwise until either the solid dissolves or you&#39;ve added all 10 mL.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Let the solution boil for at least a minute after each milliliter before adding more water.<strong> Note:</strong> It&#39;s important to add water slowly so that you don&#39;t cool the solution too much or add more water than you need.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">If you still see some solid after that, those are impurities that you must remove by hot filtration. If you do not see solid, continue the lab at step 14.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">To perform hot filtration, place about 15 mL of deionized water and one or two boiling chips in a 50-mL beaker and start heating it on a hotplate.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Clamp another 125-mL Erlenmeyer flask on a hotplate and place a wide stem or stemless glass funnel in the flask. Fold a large piece of circular filter paper in quarters, place it point down in the funnel, and open it into a cone.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once the water boils, use a pipette to wet the filter paper with hot water. Then, use tongs to pick up the flask of acetanilide solution and pour the hot liquid into the funnel, being careful not to let it overflow. <strong>Note:</strong> If the stir bar falls into the funnel, retrieve it with tweezers and rinse it with hot water.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Then, rinse the inside of the first flask with a few milliliters of hot water, pour the contents into the funnel, and dissolve any crystals that form with hot water.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Once the funnel is empty, use tongs to move it to the first flask. Carefully unclamp the second flask. You can now continue with the lab as usual.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Once there is no visible solid in your hot acetanilide solution, turn off the heat and stir motor and use tongs to place the flask on the floor of the hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Leave the solution undisturbed as it cools to room temperature, which usually takes at least 15 min. Moving or shaking the flask will make it harder for crystals to form.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">While you wait, place a small amount of the starting acetanilide material in a weighing boat and bring it to the melting point apparatus.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Pack a capillary tube with the acetanilide and measure and record its melting point.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Once the solution cools, look for crystals without moving the flask too much. If you don&#39;t see any, use a glass rod to gently scratch the inside of the flask under the solution. It&#39;s easier for crystals to start to form on scratched or rough surfaces.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">If crystals still haven&#39;t formed after 10 more min, ask your instructor for a seed crystal of high purity acetanilide. Measure and record the mass of the seed crystal, and then drop it into the room temperature acetanilide solution. Crystal growth will then occur on the seed crystal.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Once you see several crystals in the flask, prepare two ice baths in 600-mL beakers. One ice bath should nearly fill the beaker, and the other should occupy about half of the beaker.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Measure 5 mL of deionized water and place it in the full ice bath. Confirm that the flask is cool to the touch and that several crystals have formed before gently placing it in the half-full ice bath. Leave the flask undisturbed to improve crystal growth.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Check the crystal growth progress every 15 min. Once it looks like most of your acetanilide has crystallized, or you don&#39;t see much crystal growth over a 15-min period, set up for a vacuum filtration using a 125- or 250-mL filter flask.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Wet the filter paper with 1 mL of ice-cold water. Then, start the vacuum and pour the contents of the flask into the B&#252;chner funnel of the vacuum filtration setup.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Scrape crystals from the flask into the funnel using a glass rod or flexible spatula and rinse the remaining crystals into the funnel with about 1 mL of cold deionized water.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Then, stop the vacuum, break the seal, and pour the rest of the cold deionized water into the funnel. Gently stir the crystals with the glass rod for a few seconds to wash them, being careful not to tear the filter paper.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Turn on the vacuum to drain the funnel. Then, stop the vacuum, break the seal, and empty the filtrate into a waste beaker. Turn the vacuum back on, and leave the washed crystals in the funnel while the vacuum pump pulls air through them until they are dry, which usually takes about half an hour. You&#39;ll start working on your next recrystallization while you wait.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Recrystallization of <em>trans</em>-Cinnamic Acid<button class="expand">Expand</button>
	<p>In the second half of the lab, you&#39;ll recrystallize <em>trans</em>-cinnamic acid, which we&#39;ll just call cinnamic acid. Cinnamic acid is soluble in ethanol and nearly insoluble in water. So, you&#39;ll use a mixture of 95% ethanol and 5% water so that cinnamic acid is still soluble at high temperatures but less soluble at low temperatures.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">To get started, fill a 5- or 10-mL graduated cylinder with deionized water and bring it to your hood.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Then, weigh 0.5 g of cinnamic acid, record its mass, and place it in a 25-mL Erlenmeyer flask.
		<h4><strong>Table 2: Recrystallization of<em> trans</em>-Cinnamic acid</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Compound</strong></td>
					<td><strong>Mass (g) </strong></td>
					<td><strong>Melting point (&#176;C) </strong></td>
				</tr>
				<tr>
					<td><em>trans</em>-Cinnamic acid</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Seed crystal (if used)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Recrystallized<em> trans</em>-cinnamic acid<br />
					<em>(subtract seed crystal if used)</em></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td><strong>Percent recovery</strong></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/58/Table_2._Recrystallization_of_trans-Cinnamic_acid.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Next, obtain 4 mL of 95% ethanol, and pour it into the flask containing cinnamic acid. Place the flask on a hotplate set to medium heat and stir the solution with a glass rod until the cinnamic acid dissolves, which usually takes 7 &#8211; 10 min.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Then, turn off the heat and use a Pasteur pipette to add 5 drops of deionized water to the solution. This reduces cinnamic acid solubility. <strong>Note:</strong> If you don&#39;t see precipitation, add up to 5 more drops of water one by one. Stop adding water if you see solids starting to form.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Then, remove the flask from the hotplate and let it cool to room temperature undisturbed.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">While you wait, get a small amount of the starting cinnamic acid and measure its melting point.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Once the flask has cooled to room temperature, inspect it for crystals. If you don&#39;t see any, gently scratch the inside of the flask under the solution with the glass rod and wait for 10 min. <strong>Note:</strong> If that still doesn&#39;t work, get a seed crystal of cinnamic acid from your instructor. Remember to weigh the seed crystal and record its mass before you add it to your solution.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Once the flask is cool to the touch and contains several crystals, remake the ice baths with one ice bath shallow enough that the 25-mL flask will not be completely submerged.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Refill the graduated cylinder with deionized water. Place the flask of cinnamic acid crystals in the shallow ice bath and the water-filled graduated cylinder in the other ice bath. Let the crystals grow for at least 15 min.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Your acetanilide crystals should be dry by now, so measure their mass and melting point while you wait for your cinnamic acid to finish crystallizing. Turn off the vacuum and break the vacuum seal.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Gently place the B&#252;chner funnel in a clean beaker and bring it to the balance. Measure and record the mass of your pure acetanilide crystals. Remember to subtract the mass of the seed crystal if you used one.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Then, bring your crystals to the melting point apparatus and measure and record the melting point.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Now, place a clean, dry B&#252;chner funnel with a new piece of filter paper in the filter flask and check on your cinnamic acid. Once it looks like most of the cinnamic acid has crystallized, wet the filter paper with cold deionized water.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Start the vacuum and pour the contents of the cinnamic acid flask into the funnel. Scrape the remaining crystals out of the flask with the glass rod and rinse the flask with about 1 mL of cold water.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Now, turn off the vacuum, break the seal, and pour the rest of the cold water into the funnel. Gently stir the crystals with the glass rod to wash them, being careful not to damage the filter paper.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Turn on the vacuum to drain the funnel. Leave the vacuum running until the cinnamic crystals are dry.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Place the B&#252;chner funnel in a beaker and bring it to the balance. Measure and record the mass of your pure cinnamic acid crystals. Remember to subtract the mass of the seed crystal if you used one. Then, measure the melting point of your recrystallized cinnamic acid.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Now, dispose of your acetanilide and cinnamic acid in the designated solid waste container. Flush the filtrate down the drain with running water and pour leftover ice and water into the sink.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Clean your glassware using your usual methods and put away your other lab equipment.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Lastly, discard your used pipettes in the glass waste and throw out used filter paper and other trash in the lab trash container.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">First, calculate the percent recovery for each compound. We expect that some of each compound stayed in solution, so these percentages will be below the reported purity of your starting material.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Now, look at the differences in melting points between the starting material and the crystals. For each compound, the less pure starting material has a lower melting point and a broader range than the recrystallized compound.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Lastly, compare your results to the literature values. The melting point of acetanilide is 114 &#176;C, and the melting point of <em>trans</em>-cinnamic acid is 133 &#176;C. The melting points of your crystals should span 1 &#8211; 2 &#176;C and should be at or just below those values.</div>
		</li>
	</ul>
	</li>
</ol>

Recrystallization of Acetanilide and trans-Cinnamic Acid - Procedure

Rekrystalizacja

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Recrystallization of AcetanilideExpand
	In this part of ...

Lab Techniques

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Determining the Density of an Egg</strong><button class="expand">Expand</button>
	<p>Density is a measure of how compact a substance or object is, and it is calculated as mass divided by volume. When a dense object is placed in a liquid with a lower density, the object sinks; when the object is less dense than the liquid, it floats. When the densities are the same, the object will be suspended in the liquid. In this experiment, we&#39;ll determine the density of an egg by creating a solution with the same density. We&#39;ll measure the density of the salt solution using different volume measurement glassware and determine which is the most accurate.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-1">
		<div class="lab-step">To begin, wear the appropriate personal protective equipment, including close-toed shoes, long pants, long-sleeved shirt, lab coat, chemical splash goggles, and gloves.</div>
		</li>
		<li data-sub-note-item="1-2">
		<div class="lab-step">Measure the mass of the egg. Set a weigh boat on the balance and tare it. Carefully set the egg in the weigh boat while you record the mass in your notebook.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare the salt solution that will be used to suspend your egg. Use the 600-mL beaker and fill it 3/4 of the way full with deionized water.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Gently place your egg in the beaker, and then add about one teaspoon of NaCl into the water. Stir the solution gently until the NaCl dissolves. Continue to add NaCl until the egg floats just below the surface.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use different glassware to measure the volume of the salt solution. Weigh the 50-mL graduated cylinder on the top loading balance and record the mass. Then, measure 20 mL of your salt solution and record the volume in your notebook. <strong>Note: </strong>Place a blank sheet of paper behind the graduated cylinder to read the volume more clearly. Always view the measurement at eye level and measure the volume from the bottom of the meniscus. The lines on the graduated cylinder are 0.2 mL apart, meaning that you can read the volume to the hundredths place.
		<h4><b>Table 1: Measuring Mass</b></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Container</strong></td>
					<td style="text-align: center;"><strong>Mass of empty container (g)</strong></td>
					<td style="text-align: center;"><strong>Mass of full containter (g)</strong></td>
					<td style="text-align: center;"><strong>Mass of solution (g)</strong></td>
					<td style="text-align: center;"><strong>Percent error</strong></td>
				</tr>
				<tr>
					<td>50-mL graduated cylinder</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>10-mL volumetric pipette</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>50-mL beaker</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>20-mL volumetric flask</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/41/Table_1._Measuring_Mass.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Weigh the filled graduated cylinder and record the mass in your notebook. Then, pour the salt solution back into the 600-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use the volumetric pipette to measure volume. First, weigh the empty 50-mL beaker and record the mass in your notebook. Then, carefully measure 10 mL of the salt solution using the volumetric pipette and dispense the salt solution into the beaker.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure another 10 mL and add it to the beaker. Weigh the filled beaker and record the mass in your notebook. Then, empty, rinse, and dry the beaker.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use the volumetric flask to measure volume. Weigh the empty 20-mL volumetric flask and record the mass. Then, carefully fill the flask with the salt solution to just below the 20-mL fill mark. Do not fill it all the way. Use an eyedropper to finish filling the flask so that the bottom of the meniscus just touches the fill line on the neck of the flask. Then, weigh the full flask and record the weight.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Lastly, use the 50-mL beaker to measure volume. Weigh the empty beaker, then fill the beaker with salt solution to the 20-mL line and weigh the full beaker. Record the masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure the displacement of the egg when it is submerged in the salt solution. First, remove the egg and look at the level of the salt solution in the 600-mL beaker. <strong>Note:</strong> If it is not at a marked line, add deionized water to the solution so that the meniscus is at a readable volume and record the volume.&#160;
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td><strong>Measurements</strong></td>
					<td><strong>Salt solution using 20-mL volumetric flask&#160;</strong></td>
					<td><strong>Egg</strong></td>
				</tr>
				<tr>
					<td>Mass (g)</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td>Displacement of water (cm)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Inner diameter of 600-mL beaker (cm)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume (cm&#179;)</td>
					<td style="text-align: center;">-</td>
					<td></td>
				</tr>
				<tr>
					<td>Volume (mL)</td>
					<td style="text-align: center;">20</td>
					<td></td>
				</tr>
				<tr>
					<td>Density (g/mL)</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<strong><a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/41/Table_2._Measuring_Density.xlsx" target="_blank">Click Here to download Table 2</a> </strong></div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Gently place the egg in the beaker.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Use a ruler to measure the new height of the solution level and record the height. Then, place the ruler across the top of the beaker to measure the inner diameter.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Calculate the new volume from these measurements. Subtract the initial volume to obtain the displacement of the egg (volume).</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">To clean up from the experiment, return the egg to your instructor and pour the salt solution down the sink. Rinse all used glassware with tap water and set them out to dry. Finally, return the sodium chloride to your instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Calculate the mass of the solution in each of the different volumetric glassware by subtracting the empty glassware mass from the full glassware mass.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Keep track of uncertainty in your measurement by using significant figures. For example, when we look at the graduated cylinder measurement, the weight of the empty container is 90.92, and the weight of the full container is 111.59. The weight measurements have significant figures to the hundredths place, so it is important that significant figures are maintained throughout our calculations. Thus, the mass of the 20-mL salt solution is 20.67 g.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The 50-mL beaker weighed 28.65 g when it was empty and 47.96 g when it contained 20 mL of salt solution. Therefore, the mass of 20 mL of the salt solution is 19.31 g.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Repeat the calculation to obtain the mass of 20 mL of salt solution that was measured using the 10-mL pipette and the 200-mL volumetric flask.<b> Note:</b> Generally speaking, volumetric measurements are more accurate when using a volumetric flask.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Look at the accuracy of the other methods compared to the volumetric flask in terms of percent error. Percent error is calculated by subtracting the theoretical value from the experimental value, then dividing by the theoretical value times 100.
		<ul>
			<li><img src="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/41/41_Procedure_1b.jpg" /></li>
		</ul>
		</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the volumetric flask measurement as the theoretical value to determine the percent error for the other volume measurement methods. As expected, the volume measurement that uses the markings on the beaker is the least accurate.<strong> Note: </strong>We also know this because the known density of pure water is 1 g/mL. Thus, 20 mL of pure water weighs 20 g. The density of the salt solution must be higher than 20 g since salt was added to the water.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the density of the salt solution using the volumetric flask measurement. Divide the mass of the solution by the volume of the solution. Thus, the density of the salt solution is 1.053 g/mL.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the calculated density of the salt solution to the density of the egg. The volume calculated using the displacement of water in a beaker was found to be 61.14 cm<sup>3</sup> or 61.14 mL. The weight of the egg was measured to be 60.15 g, so its density is 0.9838 g/mL. The disparity between the density of the egg and the density of the salt solution arises from error introduced in both calculations, primarily due to volume measurement readings.</div>
		</li>
	</ul>
	</li>
</ol>

Accuracy, Precision, Uncertainty, and Significant Figures - Procedure

Techniki laboratoryjne

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Determining the Density of an EggExpand
	Density is a measure of how compact a substance or ...

Identification of Unknown Aldehydes and Ketones

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">DNPH Test for Aromatic Aldehydes and Ketones<button class="expand">Expand</button>
	<p>In this lab, you&#39;ll use the DNPH test, the Tollens&#39; test, and the iodoform test to identify two unknown aldehydes or ketones. You&#39;ll use butanone and benzaldehyde as known compounds to confirm that the tests are working as expected.</p>
	<p>You&#39;ll start the lab with the DNPH test, which relies on the reaction between 2,4-dinitrophenylhydrazine, or DNPH, and aldehydes and ketones. When you combine DNPH and an aldehyde or ketone in an acidic environment, they undergo a condensation reaction to form the corresponding hydrazone. The hydrazone then precipitates from solution. If the precipitate is red to orange, the aldehyde or ketone was aromatic. If it&#39;s orange to yellow, the aldehyde or ketone was non-aromatic or aliphatic.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on a lab coat, safety glasses, and nitrile gloves.<strong> Note: </strong>Many chemicals in this lab are toxic, corrosive, or flammable, so you will perform this lab in a fume hood. Change your gloves if you get any chemicals on them.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Get a vial of each known compound and choose two unknown compounds to analyze. Write the codes for your unknowns in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label a 250-mL beaker for aqueous waste and a 600-mL beaker for used pipettes. Put several clean Pasteur pipettes in another beaker and get more whenever you need them.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Obtain four small test tubes in a rack and label them with the four compound names, or codes, and the name of the first test.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Bring a 50-mL beaker and a graduated cylinder to the reagent hood and obtain 8 mL of the DNPH solution.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use four Pasteur pipettes to place 3 &#8211; 4 drops of each compound into its test tube. <strong>Note:</strong> Don&#39;t reuse pipettes during this lab because cross-contamination will invalidate the test results.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Add 2 mL of the DNPH solution to one of the test tubes and mix it well with a glass rod. Thoroughly rinse the glass rod with deionized water over your aqueous waste beaker to remove all traces of the ketone or aldehyde.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Add 2 mL of the DNPH solution to each of the other three test tubes in the same way. Make sure to thoroughly clean the glass rod after each one. When you&#39;re finished, let the solution sit for 15 min.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">While you wait, set up for vacuum filtration and fill a 250-mL beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">After 15 minutes, inspect the precipitates in the test tubes. <strong>Note:</strong> Benzaldehyde has an aromatic group, so you should see a red to orange solid in its test tube. Butanone is aliphatic, so you should see an orange to yellow solid in its test tube. Record the results in your lab notebook.
		<h4><strong>Table 1: DNPH, Tollens&#39;, and Iodoform Results</strong></h4>
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Compound</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>DNPH </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Tollens&#39; </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Iodoform </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Solubility in water </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Hydrazone m.p. </strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">Butanone</td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">Benzaldehyde</td>
					<td style="border: 1px solid; padding: 0px;"></td>
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				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160; &#160;</td>
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				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160; &#160;</td>
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					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/69/Table_1._DNPH,_Tollens_,_and_Iodoform_results.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Compare the solids from the unknown compounds to the known compounds. For each unknown, record whether this solid was more red-orange, like benzaldehyde, or more orange-yellow, like butanone.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Use a clean Pasteur pipette to wet the filter paper of the vacuum filtration setup with deionized water and collect the solid from one of your unknowns by vacuum filtration. Rinse out any solid left in the tube with deionized water.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Wash the solid on the filter paper with 10 mL of deionized water.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Pick up a tiny amount of the solid with a clean glass rod and smear it on pH paper. If the pH is below 6.5, rinse the solid with another 10 mL of deionized water. Keep rinsing the solid and checking its pH until it is between 6.5 and 7.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Label a small watch glass for the unknown and carefully transfer the filter paper and solid to the watch glass to dry.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Thoroughly rinse the B&#252;chner funnel with deionized water, dispose of the filtrate in the DNPH solution waste, and reassemble the vacuum filtration setup with a new piece of filter paper.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Collect and wash the solids from the second unknown compound and from the two known compounds in the same way.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Leave the four solids in your fume hood to dry (1 h).</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">The Tollens&#8217; Test<button class="expand">Expand</button>
	<p>When you combine an aldehyde with diammine silver(I), or Tollens&#39; reagent, the reagent is reduced to metallic silver and ammonia. The silver either coats the inside of the reaction vessel or precipitates as a black solid, so it&#39;s easy to see that the reaction happened.</p>
	<p>Most ketones are harder to oxidize than aldehydes and therefore don&#39;t react with the Tollens&#39; reagent. For this lab, you can assume that if silver appears, the compound is an aldehyde. If silver does not appear, the compound is a ketone.</p>
	<p>The Tollens&#39; reagent solution contains a strong base and must be disposed of specially to avoid forming an explosive decomposition product. Handle it carefully and remember to put the Tollens&#39; test waste in the designated container.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-4">
		<div class="lab-step">Place your used test tubes in a beaker and label four new tubes with the compound names or codes and Tollens&#39; test.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Bring a clean 50-mL beaker and a 5-mL graduated cylinder to the reagent hood and obtain 4 mL of Tollens&#39; reagent.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use clean pipettes to place 3 &#8211; 4 drops of each compound in its test tube.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Add 1 mL of Tollens&#39; reagent to each test tube and mix the solutions well with a clean glass rod, thoroughly rinsing the rod with deionized water between tubes. Set the rack of tubes to the side and let it sit for 10 min.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">While you wait, get about 100 mL of water in a 150-mL beaker and set it on a hotplate. Position a small 3-prong clamp above the beaker.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Check the benzaldehyde tube after 10 min.<strong> Note:</strong> Benzaldehyde should have a positive result, as indicated by a silver film or precipitate. If you don&#39;t see a positive result in the benzaldehyde tube, secure a thermometer to the test tube and heat in the water bath at 35 &#176;C for 5 min. The silver film, or precipitate, should then appear. Record this result in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Butanone won&#39;t react with Tollens&#39; reagent. <strong>Note: </strong>The butanone tube is an example of a negative Tollens&#39; test result. Record this in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Check the tubes of your unknowns. If you see a silver mirror or precipitate, record a positive Tollens&#39; test result for that unknown. If you don&#39;t, heat the tube in the water bath for 5 min and check again. If the test is still negative, record that result in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">When you&#39;re done, turn off the hotplate and dispose of the Tollens&#39; test solutions in the appropriate waste container. Put silver-coated tubes in the collection box for acid cleaning and put the rest in your beaker.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Iodoform Test for Methyl Ketones<button class="expand">Expand</button>
	<p>When you combine iodine and a methyl ketone in aqueous base, iodine replaces the alpha methyl&#39;s hydrogens. An addition-elimination reaction followed by proton transfer yields iodoform and a carboxylate anion. Iodoform is insoluble in water, so you&#39;ll see a yellow precipitate if the compound that you&#39;re testing is a methyl ketone. If it is not a methyl ketone, the test solution will eventually turn brown because of the unreacted iodine.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Before the iodoform test, check the solubility of all four compounds in water. Label four clean test tubes with the compound names or codes and H<sub>2</sub>O. Label another four test tubes with the compound names and CHI<sub>3</sub>.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Use clean Pasteur pipettes to place 3 &#8211; 4 drops of each compound in its test tube.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Add 2 mL of deionized water to each labeled tube. Mix well with a clean glass rod. Thoroughly clean the rod with deionized water between tubes.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Wait a few minutes for the liquids to settle before evaluating them. <strong>Note:</strong> Butanone is moderately soluble in water, so you should see little to no separation. Benzaldehyde is sparingly soluble in water, and its density is a little higher than water&#39;s. You should see a small layer at the bottom of the tube.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Inspect your unknowns for liquid layers and record the results for all four compounds in your lab notebook.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Add 2 mL of deionized water to the butanone tube and any water-soluble ketones and mix them well with a clean glass rod. <strong>Note: </strong>If you have an unknown ketone that is insoluble in water, instead add 2 mL of 1,2-dimethoxyethane and mix it well with a clean glass rod.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Now, perform the iodoform test, which is for ketones only. Use a clean 50-mL beaker and a small graduated cylinder to obtain 4 or 6 mL of 3 M NaOH, depending on whether you have two or three ketones to test.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Pour ~10 mL of iodoform reagent into another 50-mL beaker.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">Add 2 mL of 3 M NaOH to each ketone tube and mix well, thoroughly cleaning the glass rod with deionized water between each tube.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Obtain a clean Pasteur pipette and add the iodoform reagent dropwise to the butanone tube, frequently stirring until you see a yellow precipitate. <strong>Note:</strong> Butanone is a methyl ketone, so it will produce a positive result.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Rinse the glass rod with deionized water and start adding the iodoform reagent dropwise to your first, or only, unknown ketone, while stirring frequently. Continue adding the reagent until either yellow iodoform precipitates, like you saw with butanone, or the solution stays brown, even after you stir it well. <strong>Note: </strong>The latter is a negative result.</div>
		</li>
		<li data-sub-note-item="3-13">
		<div class="lab-step">If you have another unknown ketone, clean the glass rod well and repeat the test with your second ketone.</div>
		</li>
		<li data-sub-note-item="3-14">
		<div class="lab-step">When you&#39;re finished, dispose of your iodine-containing solutions in the appropriate waste container.</div>
		</li>
		<li data-sub-note-item="3-15">
		<div class="lab-step">Once the solids from the DNPH test are dry, measure the melting point of each solid and record them in your lab notebook. Put the remaining solutes in the waste container for solid DNPH derivatives.</div>
		</li>
		<li data-sub-note-item="3-16">
		<div class="lab-step">Dispose of the leftover organic liquids and the aqueous aldehyde mixtures in the usual waste containers.</div>
		</li>
		<li data-sub-note-item="3-17">
		<div class="lab-step">Rinse the Pasteur pipettes used for the aldehydes and ketones with acetone before putting them in the glass waste. Pour your aqueous waste down the drain with running tap water. Clean your glassware, put away your lab equipment, and collect and throw out any trash in your fume hood before you leave.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="4-1">
		<div class="lab-step">Identify your unknown compounds. There are six options: acetophenone, butanal, 3-methyl-2-butanone, pentanal, 2-pentanone, and 3-pentanone.</div>
		</li>
		<li data-sub-note-item="4-2">
		<div class="lab-step">Look at the DNPH results. If the precipitate was red to orange, the unknown aldehyde or ketone is aromatic. If it was yellow, the unknown is aliphatic. <strong>Note:</strong> Since only one possible compound is aromatic, red precipitate means that the unknown was acetophenone. As confirmation, you should also have seen a negative Tollens&#39; test and a positive iodoform test.</div>
		</li>
		<li data-sub-note-item="4-3">
		<div class="lab-step">A yellow precipitate in the DNPH test means that the unknown could be any of the other five compounds.</div>
		</li>
		<li data-sub-note-item="4-4">
		<div class="lab-step">Look at the results of the Tollens&#39; test. If you saw a silver mirror or silver-gray precipitate in the test tube, the unknown compound is an aldehyde. If you didn&#39;t see any change, then the unknown compound is a ketone. If your unknown is an aldehyde, it could be pentanal or butanal.</div>
		</li>
		<li data-sub-note-item="4-5">
		<div class="lab-step">Compare the melting point of the unknown&#39;s hydrazone derivative to literature values for each aldehyde to figure out which one it was.</div>
		</li>
		<li data-sub-note-item="4-6">
		<div class="lab-step">If Tollens&#39; test was negative and the DNPH test showed an aliphatic compound, the unknown could be 3-methyl-2-butanone, 2-pentanone, or 3-pentanone.</div>
		</li>
		<li data-sub-note-item="4-7">
		<div class="lab-step">Check the results of the iodoform test to narrow it down. If you saw solid yellow iodoform, the compound was a methyl ketone. If you saw a brown solution, it was not a methyl ketone.</div>
		</li>
		<li data-sub-note-item="4-8">
		<div class="lab-step">Only one of the aliphatic ketones is not a methyl ketone, so if you saw a brown solution, the unknown was 3-pentanone. If you saw yellow iodoform precipitate from solution and the DNPH test also had yellow precipitate, then the unknown is 3-methyl-2-butanone or 2-pentanone.</div>
		</li>
		<li data-sub-note-item="4-9">
		<div class="lab-step">Use the melting point of the unknown&#39;s hydrazone derivative to figure out which methyl ketone you had.</div>
		</li>
	</ul>
	</li>
</ol>

Test for Aldehydes and Ketones: DNPH, Tollen's Test, and Iodoform Test - Procedure

Identyfikacja nieznanych aldehydów i ketonów

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	DNPH Test for Aromatic Aldehydes and KetonesExpand
	In ...

Artificial Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing Artificial Selection in Plants
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Two weeks before beginning the pollination experiments, collect a sufficient amount of small pots with a drainage hole in the bottom of each pot. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Fill each pot about halfway with moist potting soil.  NOTE: It is recommended to use a minimum of 50 plants for generation one. It is standard practice to list the species name and date of planting on each individual pot, although this is not necessary to label the generation one plants.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Add a fertilizer pellet to the surface of the soil and then add more soil to the pot, enough to cover the pellet. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Use a dissecting probe to make a shallow &#188; inch depression in the surface of the soil. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Then, touch a toothpick tip to a glue stick. Use the sticky end of the toothpick to pick up one of the Brassica seeds and place the seed into the depression in the soil.  NOTE: The dissecting probe can be used to knock the seed off the toothpick as needed.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Lightly cover the seed with a thin later of soil.  Then, repeat steps 3 &#8211; 6 for each pot.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Place the pots in a brightly lit area, such as near a south-facing window or under a grow light.  NOTE: Ensure the light is uniformly distributed across the group of plans. If this is not possible, rotate the plants every few days to ensure equal exposure.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Water the plants with a plastic dropper until a small amount drains out of the pot. Do this as frequently as necessary to keep the soil moist.
</div></li></ul></li></ol>

Artificial Selection: Visualization in Plants and Data Analysis - Prep

Sztuczna selekcja

Observing Artificial Selection in Plants
ExpandTwo weeks before beginning the pollination experiments, collect a sufficient amount of small pots with a ...

Evolution

Animal Behavior

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Making Choice Chambers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the bottom of both bottles. One line should be 1 cm from the base of the bottle and the other should be 1.5 cm from the base of the bottle.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a razor blade to make a small cut in each bottle at the mark. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, insert the scissors and carefully cut along the line until the bottoms of the bottles are removed completely.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Fit one bottle bottom into the other.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Tape the bottles together to make a secure chamber.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Use the razor blade and scissors to poke a hole into the center of the chamber. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Take a piece of tape and fold the edge onto itself to make a tab and place the tape over the hole.  Note: The tab helps with easy removal and reattachment of the tape to the chamber.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Use the Sharpie to label one side of the chamber A and the other side B.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Upon receiving the colony of <em>Drosophila</em>, perform a count to make sure there are 40 living individuals in the vial.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Geotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To perform a geotaxis assay, first attach a clean, empty bottle cap to each end of the choice chamber and turn the fly vial upside down onto its lid.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Open the tape flap in the center of the choice chamber.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Insert the funnel into the hole and tap all of the flies into the chamber. HYPOTHESES: The alternate hypothesis is that more flies will be moving against gravity, or up, regardless of the orientation of the choice chamber. Alternatively, if the flies are positively geotactic, then more flies will move down, or with gravity. The null hypothesis is that the flies will have no preference with regard to gravity and will move randomly within the chamber. 
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Once the <em>Drosophila</em> are in the chamber, close the tape flap, rotate the chamber so that it stands vertically with side A on the bottom, and start the timer.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Immediately, count the number of <em>Drosophila</em> that are moving up and the number that are moving down. Record these data for time point 0 in the table. 
</div></li><li data-sub-note-item="2-6"><div class="lab-step">After 30 seconds, rotate the chamber so that side B is on the bottom and count the number of <em>Drosophila</em> that are moving up or down. Record these data for time point 30 in the table.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">After 60 seconds, rotate the chamber so that side A is on the bottom again and record the number of moving flies for time point 60.  <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/15/Table_1_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Hold the chamber over the fly vial and carefully open the cap to return all of the <em>Drosophila</em> back to their empty vial. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Phototaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">To perform a phototaxis analysis, first cover side A of the choice chamber with tin foil, taking care that the foil reaches to the exact center between the two sides. 
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Ensure the caps are secured on both ends of the chamber.  HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found in the light side of the choice chamber, if the flues are attracted to light, or on the dark side if they are repelled by light. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus. Therefore, equal numbers of flies will be found on either side of the choice chamber.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Open the tape flap in the center of the choice chamber, insert the funnel into the hole, and tap all 40 flies into the chamber as previously described.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Set the timer to sound an alarm at 10 minutes.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">When the alarm sounds, stop the timer, and count the number of flies in the open side B of the chamber.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">Subtract that number from the total number of flies added to the choice chamber to calculate the number of flies in the covered side A.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record these data in the table for time point 10.  <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/15/Table_2_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="3-8"><div class="lab-step">Carefully, return all of the flies back to their empty vial.
</div></li></ul></li><li data-note-item="4" data-parent-collapse="">Chemotaxis
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="4-1"><div class="lab-step">HYPOTHESES: In this exercise, the alternative hypothesis is that more flies will be found on the side of the chamber with the apple cider vinegar if the flies are attracted to the odor. Or, if the flies are repelled by the vinegar, then more of them will be found on the side of the chamber presenting the odorless water. The null hypothesis is that <em>Drosophila</em> will have no preference with regard to either stimulus and will move randomly in the chamber.  Use an individual disposable plastic pipette to dispense two drops of the apple cider vinegar onto one cotton ball for side A of the chamber. 
</div></li><li data-sub-note-item="4-2"><div class="lab-step">Use an individual disposable plastic pipette to dispense two drops of the distilled water onto a cotton ball for side B of the chamber. 
</div></li><li data-sub-note-item="4-3"><div class="lab-step">Fold two pieces of tape over themselves to create individual pieces of double-sided tape and use the tape to attach each cotton ball to the inside of the single bottle cap.
</div></li><li data-sub-note-item="4-4"><div class="lab-step">Screw the bottle caps with the cotton balls onto their corresponding sides of the chamber.
</div></li><li data-sub-note-item="4-5"><div class="lab-step">Transfer all of the flies into the chamber and set the timer to sound an alarm after 10 minutes. 
</div></li><li data-sub-note-item="4-6"><div class="lab-step">When the alarm sounds, stop the timer and record the number of flies in each side of the chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/15/Table_3_Animal_behavior.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="4-7"><div class="lab-step">At the end of the final experiment, funnel the flies into a morgue tube containing 70% ethanol. 
</div></li><li data-sub-note-item="4-8"><div class="lab-step">Dismantle the choice chambers for recycling.  NOTE: Since all of the chemicals used in these experiments are lab safe, the liquids can be disposed of down the sink, the chambers rinsed, and the solids can be thrown away in the trash. 
</div></li></ul></li><li data-note-item="5" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="5-1"><div class="lab-step">Gather all of the data collected for the geotaxis, phototaxis, and chemotaxis experiments.
</div></li><li data-sub-note-item="5-2"><div class="lab-step">Plot these values as bar graphs for different conditions and different time points.
</div></li><li data-sub-note-item="5-3"><div class="lab-step">Use these data to draw inferences about the behavior of <em>drosophila melanogaster</em>. 
</div></li></ul></li></ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Procedure

Zachowanie zwierząt

Making Choice Chambers
ExpandBefore beginning the experiments, use one of the Sharpies from the lab table to trace a line around the circumference of the ...

Ecology

Animal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Form and Function of Crickets and Crayfish
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">NOTE: To observe the relationship between form and function in animals, you will be examining and dissecting a cricket and a crayfish. Each pair of students will receive one of each of these organisms. When dissecting, be sure to wear gloves, closed-toed shoes, and safety goggles. Take care when handling dissecting tools, as they may be sharp. HYPOTHESES: In this experiment, the experimental hypothesis could be that the cricket and crayfish have similar body structures because they are both arthropods, but that they will have some specific adaptations for their different habitats and lifestyles. The null hypothesis might be that the cricket and crayfish have identical adaptations and body plans.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To begin, place the cricket on its back and observe its appendages.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">In Table 1, make detailed notes about your observations and try to predict the potential function of each of the forms you observe. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/27/Table_1_Animal_Diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Do the same with the wings of the cricket if they are present.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Consider how the structure of the wings benefits the cricket. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">If the cricket does not have wings, consider the reasons behind their absence. 
</div></li><li data-sub-note-item="1-7"><div class="lab-step">If the cricket is female, it will have an ovipositor. This can be observed as a long dark tube protruding from the posterior end and it is used to deposit eggs.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Consider why the ovipositor is useful. 
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Next, flip the cricket over onto its back and observe the mouth parts under a microscope. NOTE: Use a probe to move structures when necessary.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Make a hypothesis about the size and type of prey the cricket consumes based upon your observations.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Continue to examine the external parts of the cricket, making hypotheses about their function until you have filled at least five rows in the table with your observations.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Using tweezers to hold the cricket down, carefully remove the wings and legs from the body with the scissors.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, position the body of the cricket so that its underside is facing down.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Again with the scissors, cut the thin outer exoskeleton from the anus to the head trying not to damage any of the organs underneath.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Use the tweezers to open the incision and dissecting pins to hold open the exoskeleton.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Next, use the water pitcher to carefully add water to the Petri dish until it reaches just below the top edge. This should make the exposed organs of the cricket easier to see.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Move the Petri dish to the stage of the dissecting microscope and turn on the light.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Use the cricket figure as a reference and observe the organs under the dissecting microscope. Where necessary, use the probe to move organs that may be blocking others.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Identify as many internal structures as possible and fill out the form and predicted function for at least three internal structures.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you&#39;ll observe the outer and inner structures of the crayfish. Begin with the crayfish on its back and observe the appendages.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">In Table 1, take notes about the structures you notice.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Observe the tail. Devise a hypothesis for its jointed structure. Consider how a crayfish moves in its environment.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, with the crayfish still on its back, look for the swimmerets under its tail.  NOTE: If the crayfish is male, the swimmerets nearest the legs are long extending between the legs. These are specialized reproductive structures that transfer sperm to the female. Females do not have these long structures, but instead have swimmerets that store developing embryos.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Consider the benefits of this strategy. Be sure to observe both a male and female crayfish, checking with neighboring students to identify these structures.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Next, observe the mouth parts of your crayfish using a probe.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Based on these structures, make a hypothesis about the size and type of prey you think crayfish consume.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Before beginning the dissection, be sure to fill in Table 1 with at least five external structures you observed in the crayfish along with their predicted function. With the organism lying legs down, use scissors to cut an incision in the exoskeleton from the tail to between the eyes. Take care to not disturb the organs underneath.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Next, remove the carapace as demonstrated in the video.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Open the tail and use the pins to secure the exoskeleton down to the dissecting tray exposing the organs.
</div></li><li data-sub-note-item="1-30"><div class="lab-step">Observe the organs first with the naked eye. Use the figure to identify the different body parts of the animal.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Then, if there is enough space on the stage, place the dissecting tray with the crayfish under the dissecting microscope to view these structures more closely.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">Fill out at least three rows of Table 1 with these observations and predicted functions of internal structures in the crayfish.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">To analyze the data, first separate into groups. Within your groups, compare observations from your tables.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using these observations, create class predictions that relate the observed forms to the hypothesized functions for both the cricket and the crayfish. As a group, fill out Table 2 with at least five of these predictions and discuss how you would go about testing these hypotheses. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/27/Table_2_Animal_Diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Compare the internal and external structures you observed in the cricket and crayfish. Fill out Table 3 with similarities and differences you noticed in the structures of these organisms. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/27/Table_3_Animal_Diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li></ul></li></ol>

Animal Diversity: Form and Function of Crickets and Crayfish - Procedure

Różnorodność zwierząt

Form and Function of Crickets and Crayfish
ExpandNOTE: To observe the relationship between form and function in animals, you will be examining and ...

Natural Selection

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Simulating Natural Selection
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, cut the white, black, and gray pipe cleaners into quarters, which should produce a total of 100 pieces that are 3 inches long.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, cut the green pipe cleaners in the quantities and lengths specified in the video, taking care to minimize waste. This approximates a normal distribution of lengths and should result in an additional 100 pieces total. NOTE: One 12-inch pipe cleaner should yield multiple lengths, for example, a 5-inch piece and a 7-inch piece. Use this advantage to reduce waste.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Now, cut one sheet of black and one sheet of white construction paper into approximately 3-inch strips measured along the long edge of the paper.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Tape these strips down to cover the base of a dissecting tray to create an alternating pattern of black and white strips. NOTE: In the first simulation, you will use the white, gray, and black pipe cleaner segments against a black and white background to simulate different selection pressures due to predation on a population. You will select your prey using two different strategies, eyes-open and eyes-closed. For each of these strategies, you will perform four rounds consisting of 25 selections, alternating between two partners. After each round you will restore the population back to its original size using the proportions of phenotypes present after selection.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To begin the first scenario, count out 12 white, 12 black, and 26 gray pipe cleaner segments with your partner. Hypotheses: Here the experimental hypothesis might be that different predation strategies, such as having the eyes open or closed, will result in distinct selection pressures on the populations. Further, different phenotypes may be positively selected in the alternate scenarios. The null hypothesis might be that different predation strategies will have no effect on the populations or phenotypes.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Mix the pipe cleaner segments together as best as possible. Then, sprinkle them into the papered tray. NOTE: Ideally, the pipe cleaners will be spread out. However, this may not occur due to their self-adhering nature.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Once the segments are in the tray, separate them as best as possible without moving them too far from where they landed. As few segments as possible should be touching.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Have the first partner close their eyes. When given an audible signal by the second partner, the first should open their eyes and grab the first pipe cleaner they see from the tray as quickly as possible.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Set the removed pipe cleaner to the side.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Alternating partners, repeat this process a total of 25 times (steps 8 &#8211; 9).
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Count the remaining pipe cleaners by color and record this in the data collection table for Scenario 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/23/Lab_23_Tables_1_and_2.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Tables 1 and 2</a>
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, restore the population to 50 by adding one pipe cleaner of the same color for each of the remaining pipe cleaners. Hold these to the side until they are all gathered.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then, sprinkle the pipe cleaners into the tray as previously described (steps 6-7). Repeat this process for three more rounds consisting of 25 selections each and record the counts for each of the rounds in the data collection table for Scenario 1.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Remove all pipe cleaners from the tray after the fourth round.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For Scenario 2, again count out 12 white, 12 black, and 26 gray pipe cleaner segments and sprinkle them randomly into the tray.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Follow the same protocol as in Scenario 1. However, when given the signal, the grabbing partner should keep their eyes closed. To aid in accuracy, it may help to position the hand near to the tray before grabbing.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Repeat this process 25 times, switching partner roles, and record the remaining pipe cleaner amounts in the data collection table for Scenario 2.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, restore the population to 50 based on that count. Do this for all four rounds. NOTE: In the second simulation, you will be using the eyes-open and eyes-closed predation strategies as previously described to select pipe cleaners of various lengths.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">For Scenario 3, gather all 100 of the pipe cleaners that have been cut to varying lengths to represent different phenotypes.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">One partner should hold all the pipe cleaners upright and loosely in one hand resting on the tabletop, such that the bottom end of each pipe cleaner rests against the tabletop.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, the partner that is not holding the pipe cleaners should close their eyes. When given an audible signal, the partner should open their eyes and grab the first pipe cleaner they see from their partner&#39;s hand.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Repeat this process 50 times (step 21). However, the partners should not switch roles during the round this time. The partner holding the pipe cleaners should always be the one giving the signal and never grabbing from the bunch.
</div></li><li data-sub-note-item="1-23"><div class="lab-step">After the first round consisting of 50 selections, count the remaining pipe cleaners by length. Then, record this information in the data collection table for scenario three. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/23/Lab_23_Tables_3_and_4.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Tables 3 and 4</a>
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Add one pipe cleaner of the same length as each of the remaining ones to restore the population to 100. This will likely require more pipe cleaners than were originally cut. Try to use scraps from the class to reduce waste.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Repeat this for three more rounds, alternating which partner holds or grabs each round.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">To perform Scenario 4, gather the varied-length pipe cleaners in their original proportions.
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Now repeat the grabbing process, as described in Scenario 3. However, only remove 30 pipe cleaners instead of 50.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">Divide the count at the end of each round by two for each phenotype 4 inches or below, rounding fractions down.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Then, record these values in the data recording table for Scenario 4.
</div></li><li data-sub-note-item="1-30"><div class="lab-step">Use the relative proportions of the phenotypes in the final population to restore the population to 100. When restoring the population to 100, it may be necessary to calculate relative proportions of phenotypes.
</div></li><li data-sub-note-item="1-31"><div class="lab-step">Repeat this for three more rounds, again alternating holding and grabbing the pipe cleaners.
</div></li><li data-sub-note-item="1-32"><div class="lab-step">For each scenario calculate the survival for each phenotype, that is the proportion of the phenotype surviving.
</div></li><li data-sub-note-item="1-33"><div class="lab-step">Then, calculate the relative fitness, or w, between each round. As reproductive rate does not vary in this simulation, this should always be considered equal, or 1.
</div></li><li data-sub-note-item="1-34"><div class="lab-step">Also, calculate the selection coefficients for each phenotype.
</div></li><li data-sub-note-item="1-35"><div class="lab-step">Now, use the relative frequencies of each phenotype to create histograms with appropriately labeled axes for each scenario showing the population before any selection took place and then alongside these in a different color after four rounds of selection.
</div></li><li data-sub-note-item="1-36"><div class="lab-step">Identify the type of selection taking place in each scenario and label the graphs. 
</div></li><li data-sub-note-item="1-37"><div class="lab-step">Develop a hypothesis to explain your observations. 
</div></li></ul></li></ol>

Natural Selection: Calculating Fitness and Selection - Procedure

Selekcja naturalna

Simulating Natural Selection
ExpandTo begin, cut the white, black, and gray pipe cleaners into quarters, which should produce a total of 100 pieces that ...

Macromolecules

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Identifying Macromolecules with Indicator Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">HYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an indicator dye to each test solution will result in a color change if the test solution contains the macromolecule of interest. The null hypothesis might be that the addition of an indicator dye will not result in a color change in any of the test solutions.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place 16 clean test tubes in a tube rack in four rows of four tubes and label each row A through D and each column 1 - 4.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Label each test tube with its corresponding position in the test tube rack.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">After this, stir solution A thoroughly and use a pipette to add 4 mL of solution A to each tube in Row A.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next, stir solution B until it is well mixed and use a new pipette to add 4 mL of solution B to each tube in Row B.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After stirring solution C thoroughly, add 4 mL of solution C to each tube in Row C.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then add 4 mL of soybean-derived vegetable oil to each tube in Row D.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">When all of the tubes have been filled with their respective test solutions, add 200 mL of water to a 400 mL beaker on a hotplate and set the hotplate to high.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">To perform the Benedict&#39;s Test, add 2 mL of Benedict&#39;s reagent to test tubes A1, B1, C1, and D1 while the water is warming.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Observe the initial color of the tubes.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the water begins to boil, transfer each test tube to the beaker and heat the tubes in the boiling water bath for 3 - 5 minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, record the final color of each of the tubes in the appropriate cells of the table and turn off the hotplate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/CDNSource/lm/labs/22/Table_1_Macromolecules.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To perform an Iodine-Potassium-Iodide Test, first observe the color of the solutions in the test tubes in column 2.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then, add 1 mL of iodine potassium iodide reagent to tubes A2 to D2 and record the final color change in each of the tubes.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For the Sudan IV Test, observe and record the color of the solutions in the test tubes in column 3.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add 2 - 3 drops of Sudan IV reagent to test tubes A3 through D3 and record the color change, if any, in each tube.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">For the Biuret Test, observe the color of the solutions in the test tubes in column 4.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, add 1 mL of Biuret reagent to test tubes A4 through D4 and record the final color in each of the tubes.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Using the results from the indicator tests, each of the four original solutions can be identified as a sugar, starch, lipid, or protein. Refer to the Concepts video to help you determine which tubes contain which macromolecules.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">First, look at the results from the Benedict&#39;s Test. Determine which of the test solutions contain sugar based on your results.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis and the surface area in cm indicated on the y-axis.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, look at the results from the Iodine-Potassium-Iodide Test. Determine which of the test solutions contain starch.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, review the results from the Sudan IV Test. Using these observations, determine if any of the test solutions contain lipids.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Finally, look at the results from the Biuret Test. Determine if any of the test solutions contain proteins.
</div></li></ul></li></ol>

Macromolecules: Identification Using Indicator Solutions - Procedure

Makrocząsteczek

Identifying Macromolecules with Indicator Solutions
ExpandHYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an ...