Name: dna extraction from 0.22 &mu;m sterivex filters and cesium chloride density gradient centrifugation
Uploaded: 2009-09-18T05:00:00
Description: Watch this Scientific Journal Video about DNA Extraction from 0.22 Î¼M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation at JoVE.com

We would like to thank the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund and the National Sciences and Engineering Research Council (NSERC) of Canada for supporting ongoing studies on low oxygen regions of coastal and open ocean waters. J.J.W. was supported by fellowships from NSERC and D.A.W. was supported by fellowships from NSERC, Killam and the TULA foundation funded Centre for Microbial Diversity and Evolution. S.L. was supported by the TULA foundation funded Centre for Microbial Diversity and Evolution.

Note: All reagents used in this protocol are aliquoted from lab stocks into smaller working stocks- this is very important to avoid cross contamination of your DNA samples and contamination of lab stocks. Also, when pipetting, make sure to change pipette tips for every addition to avoid cross contamination.
Part One: Cell Lysis and Digestion
<ol>
<li>Begin this protocol by thawing the Sterivex filters that contain previously concentrated planktonic bioma...

<ol>
	<li>Beja, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+BAC+or+not+to+BAC:+marine+ecogenomics.">To BAC or not to BAC: marine ecogenomics.</a> Curr Opin Biotechnol. 15 (3), 187-190 (2004).</li><li>Beja, O., Suzuki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+analysis+of+bacterial+artificial+chromosome+libraries+from+a+marine+microbial+assemblage.">Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage.</a> Environ Microbiol. 2 (5), 516-529 (2000).</li><li>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=Community+genomics+among+stratified+microbial+assemblages+in+the+oceans+interior.">Community genomics among stratified microbial assemblages in the oceans interior.</a> Science. 311, 496-503 (2006).</li><li>Stein, J. L., Marsh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+uncultivated+prokaryotes:+isolation+and+analysis+of+a+40-kilobase-pair+genome+fragment+from+a+planktonic+marine+archaeon.">Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.</a> J Bacteriol. 178 (3), 591-599 (1996).</li><li>Somerville, C. C., Knight, I. T., Straube, W. L., Colwell, R. R. <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,+rapid+method+for+the+direct+isolation+of+nucleic+acids+from+aquatic+environments.">A simple, rapid method for the direct isolation of nucleic acids from aquatic environments.</a> Appl. Environ. Microbiol. 55 (3), 548-554 (1989).</li></ol>

Depending on how many samples are to be processed, this can be a time-intensive procedure due to the two one-hour incubation steps, and the repeated washes and centrifugation steps. It is best to plan two whole days for this procedure to leave plenty of time. If there are problems getting the final extract volume in the Amicon tube to reduce down to 200-500&mu;l during DNA concentration and washing, try doing additional TE washes and centrifugations (repeat Part Three) until the appropriate volume is leftover. Also, i...

A correction was made to "<a href="/video/1352">DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation</a>". A typo in a volume's measurement was corrected in step 1.2.
The step was corrected from:
Once the filter has thawed, begin the first of two incubations: one to lyse the cells and remove RNA, and one to break down proteins. For the first incubation, add 100 &#956;l lysozyme (125 mg in 1000 &#956;l TE) and 20 ml RNase A (10 &#956;g/ml) to the filter. Reseal the filter with Parafilm and leave it to rotate in the hybridization oven at 37&#176;C for 1 hour.
to:
Once the filter has thawed, begin the first of two incubations: one to lyse the cells and remove RNA, and one to break down proteins. For the first incubation, add 100 &#956;l lysozyme (125 mg in 1000 &#956;l TE) and 20 &#956;l RNase A (10 &#956;g/ml) to the filter. Reseal the filter with Parafilm and leave it to rotate in the hybridization oven at 37&#176;C for 1 hour.

A correction was made to <a href="/video/1352">DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation</a>. A typo in a volume's measurement was corrected in step 1.2.

Erratum: DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 cc syringe (26G5/8 needle)</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309597</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe (26G5/8 needle)</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309597</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml eppendorf tubes</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>0030 125.150</td>
<td>&nbsp;</td>
<tr>
<td>15ml tubes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>CLS430791</td>
<td>&nbsp;</td>
<tr>
<td>5cc syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309603</td>
<td>&nbsp;</td>
<tr>
<td>Amicon&reg; Ultra-4 Centrifugal Filter Devices</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC801096</td>
<td>10,000 Nominal molecular weight limit</td>
</tr>
<tr>
<td>Butanol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A383-1</td>
<td>Make it water saturated (butanol:water = 1:1)</td>
</tr>
<tr>
<td>Centrifuge rotor, JA5.3 </td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>368690</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge, Avanti&reg; J-E</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>369003</td>
<td>&nbsp;</td>
<tr>
<td>Cesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4036</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform:IAA (24:1)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>25666-500ML</td>
<td>&nbsp;</td>
<tr>
<td>Ethidium Bromide (EtBr)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E1510-10ml</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E5134</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization oven</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-247-10</td>
<td>37&deg;C & 55&deg;C</td>
</tr>
<tr>
<td>Lysis Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3)</td>
</tr>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L6876-5G</td>
<td>125 mg in 1000 &mu;l TE</td>
</tr>
<tr>
<td>Microcon&reg; Ultracel YM-30 Centrifugal Filter Unit</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>42410</td>
<td>50 bp cut off</td>
</tr>
<tr>
<td>Parafilm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-374-10</td>
<td>&nbsp;</td>
<tr>
<td>Phenol:Chloroform:IAA (25:24:1)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>77617-500ML</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>19133</td>
<td>&nbsp;</td>
<tr>
<td>RNase A</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP2539-100</td>
<td>10 &mu;g/ml</td>
</tr>
<tr>
<td>Safe Imager&trade; Blue light transilluminator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S37102</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Dodecyl Sulfate (SDS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4509</td>
<td>20% SDS</td>
</tr>
<tr>
<td>Sterivex filters</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SVG010RS</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0389</td>
<td>&nbsp;</td>
<tr>
<td>Table top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5415D</td>
<td>&nbsp;</td>
<tr>
<td>TE buffer, pH 8.0</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trizma&reg; base</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge rotor, TLA 100</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>343847</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tube (7X20mm, PC)</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>343775</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tube rack</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>348302</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge, Optima&reg; Max</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about DNA Extraction from 0.22 Î¼M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation at JoVE.com

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

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 ...

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