安德鲁郎的援助是在数据的图形表示赞赏。梅特勒 - 托利多提供设备和材料，开展试点工作。

简介卯木的反应已被证明是一种方便的方式快速创建多样化的化合物库（1-3）。它涉及胺的反应，醛，羧酸和腈在甲醇中，在室温下通常。卯木的反应，经常被用来作为药理活性分子的合成的工具，我们利用它可以快速访问在寻找新的抗疟疾药物（4）化合物。据观察，卯木产品有时会从反应混合物（1,5）在纯粹的形式沉淀。这是一个非常幸运的结果以来的反应，然后可以很容易地扩展，而无需昂贵的净化程序，如色谱。这将是最有利于优化卯木没有进一步的治疗反应混合物过滤，直接获得的产品产量。为此，我们使用48槽梅特勒 - 托利多MiniBlock（6）配备过滤管。一个梅特勒 - 托利多MiniMapper（7）自动化液体处理程序是用来提供的试剂和溶剂。感兴趣的参数的浓度，溶剂组成和试剂的一些多余的。 实验 MiniMapper自动化液体处理程序编程提供下列顺序液体空滤管，在48位MiniBlock：额外的溶剂，糠（甲醇2M），甲醛（甲醇2M），BOC -甘氨酸（甲醇2M）和T - butylisocyanide（甲醇2M）。默认除了体积为100微升。如果指出，过量添加一种试剂，120微升交付使用。 MiniBlock是使用内部真空过滤，然后放置16小时前在摇床。两个洗摇动过滤关闭前，由15分钟每管加入甲醇（1毫升）。管子，然后在一个dessicator高真空下干​​燥至少30分钟。滤管的重量增加产量计算。核磁共振氢纯度为评估每种溶剂系统和集中的一个样本。运行在一式三份（8-10）的反应，平均单产在表1。 卯木产品的表征 <a href="http://www.chemspider.com/Chemical-Structure.21106059.html">叔丁基（2 - {[2 - （叔butylamino）2 -羰基- 1 -苯乙基]（2 -呋喃甲基）氨基} - 2 - oxoethyl）氨基甲酸酯类</a> ：白色固体，熔点（11）202-204 C，1H NMR（12， <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp203A11-HNMR.html">频谱</a> ）（500MHZ，δppm，氘代氯仿），1.33（S，9H），1.45（S，9H），4.21（M，2H），4.49（D，J = 18Hz，1H），4.50 （ D，J = 18Hz，1H），5.47（1H），5.60（1H），5.62（1H），5.89（S，1H），6.10（1H），7.19（1H）苯基7.21-7.37（M，5H）; 13C NMR（13， <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp02060822080933CNMR.html">频谱</a> ）（500MHZ，δppm，氘代氯仿）28.3，28.6，42.3，42.8，51.7，62.9，79.5，107.7，110.4，128.5，128.7，129.6，134.7 ，141.9，149.8，155.7，168.4 170.2; IR（14， <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/ugiproductpaper03IR.html">频谱</a> ）（Ʋmax厘米的ATR - 1）：1645,1673,1699，3331;的FAB -人力资源管理系统（15 ）（C24H33N3O5计算， <a href="http://showme.physics.drexel.edu/mirza/Exp099Chighres.JPG">频谱</a>米/于444.2498 [M + H]，获得了444.2517。） 数据分析采用单因素或双因素（与复制）分析方差分析工具，在Microsoft Excel沉淀产量数据进行了分析。 （16）对于这些变量的发现在统计学意义ANOVA分析，费舍尔的最小显着差（LSD）的测试是用来确定具体的实验设置，参展卯木沉淀产量差异显着（17）。所有的显着性检验在95％置信水平。 结果与讨论 图1总结了溶剂组成卯木沉淀产量的影响。单因素方差分析表明在一个0.2M的试剂浓度研究的四种溶剂的平均收益率的显着性差异。费舍尔的迷幻剂试验表明，的沉淀物在甲醇和乙醇中获得收益率是不的95％的信心水平（酒吧图中的溶剂的名称，标志）统计学差异;卯木沉淀的产量显着甲醇和乙醇比在乙腈溶液中，这又是明显比在THF。 <img src="/files/ftp_upload/old/942_2008_9_26_0/942_Figure-01.png" alt="图1" /> 图1：卯木产量溶剂组成的影响，总结沉淀在reageNT浓度0.2M。上市溶剂包括40％，在此浓度的甲醇。 考虑到试剂浓度的影响，表1中的数据显示，在产量急剧下降，被发现时，从0.2M，0.07M，使用甲醇，乙醇和乙腈作为溶剂;单产甲醇在0.2M和0.4中号的浓度。请注意，卯木反应一般在0.5 - 2M范围（2）运行。然而，当使用一个自动化液体处理的时候，它可能会很难启动与试剂的解决方案集中2M以上，从而将终浓度约0.5米的一个实际的上限。这种优化的研究结果表明在解释低于0.2M卯木的反应是成功还是失败的谨慎，显然是由于非线性效应在作怪。 图2总结了溶剂组成和试剂卯木沉淀的试剂浓度0.2M产量过剩的效果。这两个因素的方差分析结果表明一个显着的溶剂和试剂过剩的影响。此外，还有一个两个变量之间的统计学意义的相互作用。相互作用的详细信息被发现在图2表明，沉淀物的产量是由使用的溶剂的影响。再次注意，图中的因素，酒吧的收益率在95％置信水平统计学差异。对于甲醇和乙醇，获得最佳产量过剩的亚胺或腈，乙腈，获得显着改善的结果与胺，醛或超过腈。正如上述讨论结果见表1，沉淀物的整体收益率是低的，四氢呋喃为溶剂，只有一个亚胺过剩导致在沉淀收益率的显着改善。四个溶剂提高产量，这些不同的模式是什么导致了统计显着的相互作用效果，可能是一个不同的互动与不同溶剂的卯木产品的指标。虽然在表1中的数值计算结果表明，最好的收益率在1.2等值亚胺过剩，0.2M乙腈/甲醇和0.2M乙醇/甲醇混合物，也提供了显着的产量获得0.4M甲醇。特别要注意，所有在0.2M四氢呋喃/甲醇混合物了十分恶劣的结果。卯木反应已成功地进行了多种溶剂，包括THF（2）。请注意，这里的低收益率并不意味着卯木反应，没有考虑在四氢呋喃的混合物，只是没有形成沉淀。 <img src="/files/ftp_upload/old/942_2008_10_13_5/942_Figure-02.png" alt="图2" width="335" height="325" /> 图2：溶剂组成和试剂卯木产量过剩的效果，总结沉淀的试剂浓度0.2M。上市溶剂包括40％，在此浓度的甲醇。 图3总结了卯木产量试剂浓度和试剂过剩的影响，沉淀用甲醇为溶剂。这两个因素的方差分析结果表明一个显着的浓度和试剂过剩的影响。如上所述，沉淀试剂浓度0.2M和0.4M明显比在0.07M的产量（但不从对方）。此外，还有一个正在研究这两个变量之间的统计学意义的互动。请注意，一般在较高的试剂浓度相同的模式是多余的试剂的身份在沉淀率的提高。 <img src="/files/ftp_upload/old/942_2008_9_26_2/942_Figure-03.png" alt="图3" width="384" height="255" /> 图3：卯木产量过剩的浓度和试剂的效果总结沉淀在甲醇溶剂。 总体而言，在图2和3所描述的结果表明，有溶剂的选择，试剂浓度和试剂过剩的身份之间的相互作用，但总体来说，这些比直溶剂和浓度的影响更微妙。其他人发现了类似的结果时，在优化卯木反应（18）。 结论优化实验中发现48运行的产量最高，从0.4米甲醇与1.2当量的66％。亚胺。这是显着高于49％的产量从最初的反应在甲醇中号0.4摩尔浓度下获得的。沉淀产量重现性好，获得在这些复制的实验，让微妙的相互作用被验明正身的影响。

<ol>
	<li>Marcaccini, S., Torroba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Ugi+four-component+condensation.">The use of the Ugi four-component condensation.</a> Nature Protocols. 2, 632-63 (2007).</li><li>Domling, A., Ugi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+reactions+with+isocyanides+Angew.">Multicomponent reactions with isocyanides Angew.</a> Chem. Int. Eng. Ed. 39, 3168-31 (2000).</li><li>Domling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Developments+in+Isocyanide+Based+Multi-Component+Reactions+in.">Recent Developments in Isocyanide Based Multi-Component Reactions in.</a> Applied Chemistry, Chem. Rev.. 106, (2006).</li><li>Bradley, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> http://usefulchem.blogspot.com/2008/01/we-have-anti-malarial-activity.html. , .</li><li>Sanudo, M., Marcaccini, M., Basurto, S., Torroba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+3-Hydroxy-6-oxo[1,2,4]triazin-1-yl+Alaninamides,+a+New+Class+of+Cyclic+Dipeptidyl.">Synthesis of 3-Hydroxy-6-oxo[1,2,4]triazin-1-yl Alaninamides, a New Class of Cyclic Dipeptidyl.</a> Ureas J. Org. Chem.. 71, 4578-45 (2006).</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> UsefulChem EXP201 http://usefulchem.wikispaces.com/EXP201. , .</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EXP202 http://usefulchem.wikispaces.com/EXP202. , .</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EXP203 http://usefulchem.wikispaces.com/EXP203. , .</li><li>p, m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>Bradley, J. -. C., NMR, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> spectrum 203A11 from UsefulChem EXP203 http://usefulchem.wikispaces.com/EXP203. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C NMR spectrum 206A from UsefulChem EXP206 http://usefulchem.wikispaces.com/EXP206. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IR spectrum from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FAB-MS from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis details available at http://usefulchem.wikispaces.com/space/showimage/EXP201-203_KO-A.xls. , .</li><li>Montgomery, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Analysis+of+Experiments.">Design and Analysis of Experiments.</a> Nature Protocols. , (2005).</li><li>Tye, H., Whittaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Design+of+Experiments+approach+for+the+optimisation+of+a+microwave+assisted+Ugi+reaction.">Use of a Design of Experiments approach for the optimisation of a microwave assisted Ugi reaction.</a> Org. Biomol. Chem.. 2, 813-813 (2004).</li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Furfurylamine</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F20009</td>
<td>&nbsp;</td>
<tr>
<td>Benzaldehyde</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>12010</td>
<td>&nbsp;</td>
<tr>
<td>Boc-glycine</td>
<td>Reagent</td>
<td>TCI America</td>
<td>B1185</td>
<td>&nbsp;</td>
<tr>
<td>t-butylisocyanide</td>
<td>Reagent</td>
<td>TCI America</td>
<td>B1274</td>
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optimization of the ugi reaction using parallel synthesis and automated liquid handling

描述了卯木糠，BOC -甘氨酸，苯甲醛和T - butylisocyanide混合反应的优化。一式三份运行48个平行实验的报道，不同浓度，溶剂和试剂的一些多余的。该产品的隔离是通过一个简单的过滤和洗涤程序。获取最高产量为66％，从0.4与1.2当量的中号甲醇。亚胺。这是显着高于49％的产量从最初的反应在甲醇中号0.4摩尔浓度下获得的。 0.4M或0.2M试剂浓度的甲醇溶液了优越的产量，而在0.07M所有溶剂系统表现不佳。 0.2M，甲醇和乙醇/甲醇混合物（60/40），统计学同样良好的四氢呋喃/甲醇（60/40），而穷人和乙腈/甲醇（60/40）是中间。沉淀产量重现性好，获得在这些复制的实验，让微妙的相互作用被验明正身的影响。

Watch this Scientific Journal Video about Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling at JoVE.com

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

卯木的反应已被证明是一种方便的方式快速创建不同化合物库。它涉及胺的反应，醛，羧酸和腈在甲醇中，在室温下通常。在这段视频中，我们利用配备过滤管和一个梅特勒 - 托利多MiniMapper自动化液体处理程序是用来提供的试剂和溶剂48槽梅特勒 - 托利多MiniBlock。感兴趣的参数的浓度，溶剂组成和试剂的一些多余的。

卯木反应的优化使用平行合成和自动化液体处理

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

optimization-ugi-reaction-using-parallel-synthesis-automated-liquid

Nanyang Technological University

Research

JoVE Journal

Biology

16.3K 次观看.  Drexel University. 卯木的反应已被证明是一种方便的方式快速创建不同化合物库。它涉及胺的反应，醛，羧酸和腈在甲醇中，在室温下通常。在这段视频中，我们利用配备过滤管和一个梅特勒 - 托利多MiniMapper自动化液体处理程序是用来提供的试剂和溶剂48槽梅特勒 - 托利多MiniBlock。感兴趣的参数的浓度，溶剂组成和试剂的一些多余的。

视频: Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

测定哺乳动物细胞计数，细胞的大小和使用末席ž迷你自动细胞计数细胞的健康

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

聚合酶链反应:基本协议加上故障排除和优化策略

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals across the landscape is of primary importance in watershed management. Traditional economics methods of finding the lowest-cost solution in the watershed context (e.g.,5,12,20) assume that off-site impacts can be accurately described as a proportion of on-site pollution generated. Such approaches are unlikely to be representative of the actual pollution process in a watershed, where the impacts of polluting sources are often determined by complex biophysical processes. The use of modern physically-based, spatially distributed hydrologic simulation models allows for a greater degree of realism in terms of process representation but requires a development of a simulation-optimization framework where the model becomes an integral part of optimization.
Evolutionary algorithms appear to be a particularly useful optimization tool, able to deal with the combinatorial nature of a watershed simulation-optimization problem and allowing the use of the full water quality model. Evolutionary algorithms treat a particular spatial allocation of conservation practices in a watershed as a candidate solution and utilize sets (populations) of candidate solutions iteratively applying stochastic operators of selection, recombination, and mutation to find improvements with respect to the optimization objectives. The optimization objectives in this case are to minimize nonpoint-source pollution in the watershed, simultaneously minimizing the cost of conservation practices. A recent and expanding set of research is attempting to use similar methods and integrates water quality models with broadly defined evolutionary optimization methods3,4,9,10,13-15,17-19,22,23,25. In this application, we demonstrate a program which follows Rabotyagov et al.'s approach and integrates a modern and commonly used SWAT water quality model7 with a multiobjective evolutionary algorithm SPEA226, and user-specified set of conservation practices and their costs to search for the complete tradeoff frontiers between costs of conservation practices and user-specified water quality objectives. The frontiers quantify the tradeoffs faced by the watershed managers by presenting the full range of costs associated with various water quality improvement goals. The program allows for a selection of watershed configurations achieving specified water quality improvement goals and a production of maps of optimized placement of conservation practices.

This work demonstrates an integration of a water quality model with an optimization component utilizing evolutionary algorithms to solve for optimal (lowest-cost) placement of agricultural conservation practices for a specified set of water quality improvement objectives. The solutions are generated using a multi-objective approach, allowing for explicit quantification of tradeoffs.

空间使用SWAT模型的农业保护的做法和多目标优化的进化算法

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

自动定量分析细胞计数程序使用ImageJ插件

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

全自动毛细 Electrophoretic-based 免疫分析方法的程序和关键优化策略

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

吸收荧光、磁性纳米微粒测定昆虫的吸液能力

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

使用自动成像系统测量 iPSC 的汇合度。

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...