This work was funded by the National Science Foundation Environmental Genomics Program (Grant No. EF-0723707).

1. Microbial Cell Extraction:
<ol>
<li>Homogenize microbial mats with a sterile grinding pestle by mixing thoroughly. Place approximately all 30 g (wet weight) of homogenized mat material into sterile container of Waring blender, add about 100 mL of 1 M NaCl (or a concentration specific to sample in use), and blend three times at medium speed for 1 min with intermittent cooling in a -20&deg;C freezer for 1 min. Transfer the slurry into a 250 mL centrifuge bottle and fill the remaining empty volume with 1 M NaCl. Prepare NaCl solution in autoclaved DI water and filter sterilize it.</li> 
<li>Further dislodge the microbial cells from the matrix by shaking (150 rpm) using a vortexer (Vortex-GenieR2) at room temperature for 30 min.</li> 
<li>Centrifuge at low speed (500x g) for 15 min at 4&deg;C. Gently transfer the supernatant into a flask with minimum disturbance to the sediment.</li>
<li>Using the pelleted sediment, repeat blending and steps 1.2 and 1.3 four additional times with the addition of fresh NaCl to each sediment pellet at the end of step 1.3. The supernatant from each NaCl extraction is transferred into a fresh flask.</li>
<li>Combine the supernatants from the 5 cell extractions and centrifuge in aliquots of 200 mL at high speed (25,000x g) for 15 min at 4&deg;C. Discard the supernatant, resuspend each cell pellet in 10 mL of 2% sodium hexametaphosphate, combine pellets and make up the volume to 200 mL with 2% sodium hexametaphosphate. Wash cells by shaking (150 rpm) at room temperature for 30 min. At the end of this step you should have only one tube of cells to proceed to the next step.</li>
<li>Centrifuge at high speed (25,000x g) for 15 min at 4&deg;C. Discard the supernatant, resuspend cell pellet in 200 mL of TE (50 mM EDTA), and wash cells by shaking (150 rpm) at room temperature for 10 min.</li> 
<li>Centrifuge at high speed (25,000x g) for 15 min at 4&deg;C, discard the supernatant, resuspend in 15 mL TE (10 mM EDTA), and store 200-&mu;L aliquots at -80&deg;C until needed for DNA extractions.</li>
</ol>
2. DNA Extraction and Purification:
<ol>
<li>Add 200 &mu;L 5 M NaCl, 200 &mu;L 10% SDS, and 100 &mu;L 14.3 M &beta;-mercaptoethanol to the 200-&mu;L microbial cell aliquot. Mix gently by inverting 4-6 times.</li> 
<li>Subject the mixture to 3 rounds of freeze-thaw by submerging in liquid nitrogen for 2 min followed by thawing in a 65&deg;C water bath for 5 min. Extend final thawing to 10 min.</li> 
<li>Add 200 &mu;L 5 M potassium acetate (pH 5.5) and place on ice for 10 min. Centrifuge at 10,000x g for 10 min at 4&deg;C and transfer the supernatant to a new tube using a wide-bore pipette tip. Add 3 &mu;L of RNase A and incubate at 37&deg;C for 1hr.</li>
<li>Add equal volume of chloroform, shake briefly by inversion, and centrifuge at 15,000x g for 10 min at room temperature.</li> 
<li>Carefully transfer the top layer of supernatant into a new tube using a wide-bore pipette tip. Avoid pipetting the white interface layer located between the top and bottom layers. Repeat the chloroform-extraction procedure.</li>
<li>Add an equal volume of chilled (at -20&deg;C) isopropanol to the supernatant and incubate on ice for 30 min to precipitate DNA.</li>
<li>Centrifuge at max speed (20,800x g) for 10 min at 4&deg;C to pellet DNA.</li>
<li>Discard the supernatant and wash DNA pellet with 1 mL of chilled (at -20&deg;C) 70% ethanol.</li>
<li>Centrifuge at max speed (20,800x g) for 10 min at 4&deg;C, discard the supernatant, repeat 70% ethanol wash, and air-dry DNA pellet for 10 min. Repeat 70% ethanol wash.</li>
<li>Resuspend DNA in 25 &mu;L TE (10 mM EDTA), warm at 65&deg;C for 5 min, combine into one tube if multiple tubes have been used, and make-up volume to 500 &mu;L with TE.</li>
<li>Add 500 &mu;L ice-cold (or at 4&deg;C) 20% polyethylene glycol (PEG) (prepared in 1.2 M NaCl). Mix gently by inversion, incubate on ice for 10 min, and centrifuge at maximum speed for 10 min at 4&deg;C to pellet DNA. Remove and discard the supernatant and wash the pelleted DNA with 1 mL of chilled 70% ethanol, air-dry for 10 min, and resuspend in 30-50 &mu;L TE or molecular grade water. Facilitate resuspension of DNA by warming at 65&deg;C for 5 min and Store DNA at -80&deg;C.</li> 
</ol>
3. DNA Purity, Concentration and Size Determination:
<ol>
<li>Determine DNA quality by measuring the A260/280 and A260/230 ratios using a Nanodrop 1000 spectrophotometer or any other appropriate instrumentation.</li>
<li>Determine DNA concentration using Quant-iT dsDNA Assay kit according to the manufacturer&#x2019;s instructions.</li>
<li>Determine DNA size using pulse field gel electrophoresis. Prepare 1% agarose gel with 0.5X TBE and use the following parameters on the CHEF Mapper XA System; initial switch time of 0.35 s, a final switch time of 7.67 s, and 120&deg; included angle for a gradient of 6.0 V/cm at a linear ramping factor. Stain gel with 1 x SYBR Green I for 30 min. Load between 700-1000 ng total DNA on gel. See Fig. 3 for a condensed form of the complete protocol.</li>
</ol>
Representative Results:
Cell extraction:
Sequential microbial cell extracts (supernatants) have shown that the turbidity of extracts decreases as the number of extractions increase. This suggests a reduction in the number of cells following each successive cell extraction. Important to note here is that as each additional cell extraction step provides the opportunity for contaminant introduction, the number of cell extractions should be minimized so that the final cell pellet is representative of the overall microbial community. Other sources of contamination were controlled by adopting adequate laboratory sterile techniques. For instance, the solutions were prepared in autoclaved DI water and filter sterilized. Containers were sterilized with alcohol, autoclaved, and treated under UV light and ultraviolet crosslinker. 
DNA concentration and quality determination:
The protocol yielded approximately 2 &mu;g of HMW DNA (35-50 kb) (Fig. 4) per gram of mat sample with an A260/280 ratio of 1.6, and an A260/230 ratio of 0.7 (Table 1). Although the A260/230 ratio appeared to be low, no inhibition of downstream molecular-based application such as PCR-amplification of the 16S rRNA genes was observed in a separate study7. It is important to note that DNA from hypersaline mats has two main sources of contamination; EPS and salts. It is therefore possible that traces of these contaminants may be influencing the A260/230 ratios despite the tremendous efforts to reduce their effects on down stream applications.
DNA size determination:
Pulse field gel electrophoresis employs a pulsating current flow with intermittent directional switches resulting in a DNA smear as shown in figure 3. Our protocol yielded a HMW DNA of approximately 35-50 kb. While some DNA size may be smaller than 30 kb, it is important to have part of the smear above 35 kb given that fosmid cloning requires &tilde;40 kb DNA inserts and larger DNA fragments provide greater access to intact biosynthetic pathways. In our studies, the DNA smear above 35 kb was excised and purified for large-insert vector cloning and other molecular applications.
<img src="/files/ftp_upload/2887/2887fig1.jpg" alt="Figure 1"/> 
Figure 1. Hypersaline microbial mat sampling site (Big Pond) located on Eleuthera, The Bahamas.
<img src="/files/ftp_upload/2887/2887fig2.jpg" alt="Figure 2"/> 
Figure 2. A cross-section of the hypersaline microbial mat used in this study. The microbial mat was obtained from a hypersaline pond located on Eleuthera, The Bahamas.
<img src="/files/ftp_upload/2887/2887fig3.jpg" alt="Figure 3"/> 
Figure 3. Schematic representation of procedures involved in microbial cell extraction, cell lysis, extraction, and purification of metagenomic DNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2887/2887fig3large.jpg">Click here to view a larger image.</a>
<img src="/files/ftp_upload/2887/2887fig4.jpg" alt="Figure 4"/> 
Figure 4. Molecular weight characterization of extracted DNA using pulse field gel electrophoresis. Lane M is a HMW marker, and lanes 1 and 2 are replicates of metagenomic DNA extracted from the Eleuthera hypersaline mat using the above protocol.
<table border="1">
	<tbody>
		<tr>
			<td>Extraction Method</td>
			<td>&nbsp;</td>
			<td>Concentration ng/g</td>
			<td>A260/280</td>
			<td>A260/230</td>
		</tr>
		<tr>
			<td>PEG</td>
			<td>Rep 1</td>
			<td>1806.1</td>
			<td>1.64</td>
			<td>0.76</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Rep 2</td>
			<td>2010.8</td>
			<td>1.61</td>
			<td>0.75</td>
		</tr>
	</tbody>
</table>
Table 1. Measurement of concentration and quality of DNA extracted from microbial hypersaline mat.

<ol>
	<li>Decho, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms+in+intertidal+systems:+an+overview.">Microbial biofilms in intertidal systems: an overview.</a> Cont. Shelf Res. 20, 1257-1273 (2000).</li><li>Dupraz, C., Visscher, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+lithification+in+marine+stromatolites+and+hypersaline+mats.">Microbial lithification in marine stromatolites and hypersaline mats.</a> Trends Microbiol. 13, 429-438 (2005).</li><li>Steffan, R. J., Goksoyr, J., Boj, A. K., Atlas, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+from+soils+and+sediments.">Recovery of DNA from soils and sediments.</a> Appl. Environ. Microbiol. 54, 2908-2915 (1988).</li><li>Lee, Y. K., Kim, H. W., Liu, C. L., Lee, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+DNA+extraction+from+marine+bacteria+that+produce+extracellular+materials.">A simple method for DNA extraction from marine bacteria that produce extracellular materials.</a> J. Microbiol. Methods. 52, 245-250 (2003).</li><li>Roose-Amsaleg, C. L., Garnier-Sillam, E., Harry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+Purification+of+Microbial+DNA+from+Soil+and+Sediment+Samples.">Extraction and Purification of Microbial DNA from Soil and Sediment Samples.</a> Appl. Soil Ecol. 18, 47-60 (2001).</li><li>de Lipthay, J. R., Enzinger, C., Johnsen, K., Aamand, J., S&#248;rensen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+DNA+extraction+method+on+bacterial+community+composition+measured+by+denaturing+gradient+gel+electrophoresis.">Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis.</a> Soil Biol. Biochem. 36, 1607-1614 (2004).</li><li>Bey, B. S., Fichot, E. B., Dayama, G., Decho, A. W., Norman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+High+Molecular+Weight+DNA+from+Microbial+Mats.">Extraction of High Molecular Weight DNA from Microbial Mats.</a> Biotechniques. 49, 631-640 (2010).</li><li>Kakirde, S. K., Parsley, L. C., Liles, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+does+matter:+Application-driven+approaches+for+soil+metagenomics.">Size does matter: Application-driven approaches for soil metagenomics.</a> Soil Biology &#38; Biochemistry. 42, 1911-1923 (2010).</li><li>Rodon, M. R., August, P. R., Bettermann, A. D., Brady, S. F., Grossman, T. H., Liles, M. R., Loiacono, K. A., Lynch, B. A., MacNeil, I. A., Minor, C., Tiong, C. L., Gilman, M., Osburne, M. S., Clardy, J., Handelsman, J., Goodman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+the+Soil+Metagenomic:+A+Strategy+for+Accessing+the+Genetic+and+Functional+Diversity+of+Uncultured+Microorganisms.">Cloning the Soil Metagenomic: A Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms.</a> Appl. Environ. Microbiol. 66, 2541-2547 (2000).</li><li>Beja, O., Aravind, L., Koonin, E. V., Suzuki, M. T., Hadd, A., Nguyen, L. P., Jovanovich, S. B., Gates, C. M., Feldman, R. A., Spudich, J. L., Spudich, E. N., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Rhodopsin:+Evidence+for+a+New+Type+of+Phototrophy+in+the+Sea.">Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea.</a> Science. 289, 1902-1906 (2000).</li></ol>

No conflicts of interest declared.

Given that total cell removal from complex and highly diverse microbial mat samples is not practical, the primary concern is how well the extracted cells represent the overall microbial mat community. In a previous study, PCR-DGGE analysis of microbial 16S rRNA genes showed that the five cell removal steps used in this protocol extracts cells that are representative of the overall microbial mat community7. The actual number of cell extraction steps required to provide a cell pellet that is representative of the overall microbial community will likely change dependent upon sample type. For optimal protocol development for different samples, a small-scale pilot study is recommended wherein empirical testing of the microbial richness recovered after each cell extraction step is compared to the richness of the original community.
Fosmid cloning requires HWM DNA of 35-40 kb for clone library construction8. Our protocol yielded DNA with sizes ranging from 35-50 kb (Fig. 4) and was successfully used to generate a fosmid-based metagenomic library (unpublished results). Other protocols used in DNA extraction from EPS-producing marine bacteria yielded &tilde;23 kb4. In functional metagenomic studies, large DNA fragments provide greater access to a wide range of genes encoding for biosynthetic pathways as well as for functional behaviors 9,10. Thus, HMW DNA is an important requirement for metagenomic studies. Generating large-insert metagenomic libraries from environmental samples will increase the chances of discovering novel biosynthetic pathways that may lead to the discovery of novel genes. This protocol may be used to extract DNA from other refractory environmental samples.

<table border="1">
	<tbody>
		<tr>
			<th>Name of reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polyethylene glycol 8000</td>
			<td>Promega</td>
			<td>V3011</td>
			<td>20% in 1.2 M NaCl</td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr>
			<td>Quant-iT dsDNA Assay kit</td>
			<td>Invitrogen</td>
			<td>Q33130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase</td>
			<td>Epicentre </td>
			<td>MRNA092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>BDH Chemicals</td>
			<td>BDH8014</td>
			<td>Appropriate conc.</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Fisher Scientific</td>
			<td>03-500-509</td>
			<td>10% in water</td>
		</tr>
		<tr>
			<td>sodium hexametaphosphate</td>
			<td>EMD Chemicals</td>
			<td>SX0583-3</td>
			<td>2% in water</td>
		</tr>
		<tr>
			<td>TBE</td>
			<td>Fisher Scientific</td>
			<td>BP1333-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CHEF Mapper XA System</td>
			<td>Bio-Rad Laboratories</td>
			<td>170-3670</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NanoDrop 1000 spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Scientific Industries Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultraviolet Crosslinker</td>
			<td>UVP</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Waring blender</td>
			<td>Waring laboratory </td>
			<td>LB10S</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

extraction of high molecular weight dna from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

Watch this Scientific Journal Video about Extraction of High Molecular Weight DNA from Microbial Mats at JoVE.com

Extraction of High Molecular Weight DNA from Microbial Mats

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

extraction-of-high-molecular-weight-dna-from-microbial-mats

Nanyang Technological University

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JoVE Journal

Biology

19.5K Views.  Arnold School of Public Health, University of South Carolina. We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Video: Extraction of High Molecular Weight DNA from Microbial Mats

Biology of Microbial Communities  - Interview

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

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.

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

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA. We also describe how sedimentation velocity experiments in the analytical ultracentrifuge, and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...