Name: DNA Extraction from 0.22 and mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation (Video) | JoVE Journal
Uploaded: 2009-09-18T05:00:00.000+00:00
Duration: 15 min 14 s
Description: Learn DNA extraction from Sterivex filters and cesium chloride purification for environmental DNA studies. This method enables high-quality genomic DNA recovery from marine planktonic biomass.

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5 minutes

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 biomass on ice. For simplicity, here we describe the procedure for processing an individual filter. In practice, we recommend processing 16 or fewer filters at a time. </li>
 <li>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 μl lysozyme (125 mg in 1000 μl TE) and 20 ml RNase A (10 μg/ml) to the filter. Reseal the filter with Parafilm and leave it to rotate in the hybridization oven at 37°C for 1 hour.</li>
 <li>For the next incubation, add 100 μl Proteinase K and 100 μl 20% SDS to the filter. Reseal using Parafilm and leave it to rotate one to two hours more in the hybridization oven, this time at 55°C. </li>
 <li>Using a 5cc syringe, transfer the lysate from the Sterivex filter into a 15 ml Falcon tube. Rinse the filter with 1 ml lysis buffer and add the rinse liquid to the lysate in your Falcon tube using the 5cc syringe. Now that you have lysed and digested your cells, you are ready to extract their DNA. </li> 
 </ol>
Part Two: DNA Extraction
<ol>
<li>The next step is to use the phenol-chloroform method to extract the DNA from your lysate. Add an equal volume (about 3ml) of Phenol:Chloroform:Isoamyl Alcohol (IAA) to the lysate tube, making sure to change pipette tips for each addition to avoid cross contamination. Vortex for 10 seconds to mix well.</li>
 <li>Spin the tube at 2500 g for 5 minutes, making sure the centrifuge is balanced. During centrifugation, the phenol-chloroform-lysate mixture should separate into two layers: an organic layer containing the phenol, chloroform, and proteins from your sample, on the bottom, and an aqueous layer, which consists of your DNA, water, and other more hydrophilic molecules, on top.</li>
 <li>Transfer the aqueous layer (containing your DNA) into a new 15 ml Falcon tube. Be careful not to touch the interface with the pipette tip (always leave a small amount of the aqueous layer behind).</li>
 <li>To this new tube, add an equal volume (approx 3mL) of Chloroform:IAA, making sure to change pipette tips for each addition to avoid cross contamination. Vortex for 10 seconds. This step helps to remove any remaining phenol from your DNA sample. </li>
 <li>Spin at 2500 g for 5 minutes or until aqueous layer is clear. Transfer aqueous layer into a new, labeled Falcon tube and add 1 ml of TE at pH 8.0, being careful not to touch the interface with the pipette tip (always leave a small amount of the aqueous layer behind). This aqueous layer contains your DNA extract.</li>
 </ol>
Part Three: DNA Concentration and Washing
<ol>
<li>The next step is to wash and concentrate the DNA sample in a 15 ml Amicon Ultra centrifuge tube. The Amicon tubes consist of a filter that retains molecules at or above a specified molecular weight (the retentate) and a tube to catch the flow-through (the filtrate). In this case, the filter retains your DNA (the retentate). Transfer your DNA extract from step 2.5 to the filter compartment of an Amicon Ultra tube. </li>
 <li>Spin at 3500 g for 10 minutes. Check to make sure there is less than 1 ml of liquid retained in the Amicon filter. If more retentate remains, refill the filter with TE and spin again. Make sure to use a fresh pipette tip for every TE addition to avoid cross contamination.</li>
 <li>Remove the flow-through, or filtrate, to another Falcon tube and save it in the fridge until you have confirmed your final DNA product on a gel (Part Four). </li>
 <li>Add 2 ml TE buffer to the Amicon filter and spin at 3500 g for 6 minutes, making sure to use a fresh pipette tip for each TE addition to avoid cross contamination. Pool filtrate with that from step 3.3 and save in the fridge. </li>
 <li>Wash the DNA twice more with TE for a total of three washes (making sure to always use a fresh pipette tip), and saving the filtrate each time (as per steps 3.3 and 3.4). For the last wash, spin until 200 to 500 ml of retentate remains in the Amicon filter. Record the final volume and transfer the retentate (containing your DNA) to a labeled 1.5 ml Eppendorf tube. </li>
</ol>
Part Four: Determination of DNA Quality
<ol>
<li>Check the DNA quality and estimate the concentration of the DNA by running out your samples on a gel 0.8% agarose gel stained with 1ml ethidium bromide (EtBr) overnight. (Be extremely cautious when using EtBr- it is a known mutagen and suspected carcinogen. Make sure to change gloves often and avoid EtBr transfer to the rest of the lab and to your skin.) Load the samples next to DNA ladders, which serve as size and intensity standards (see next step). </li>
 <li>In the first several lanes, load ladders with bands of various molecular weights and concentrations. We recommend the following loading pattern: in the first and last lanes (outermost lanes), load 10 μl of 50ng/μl of 1kb+ or 2log ladder. In lanes 2,3 and 4, load 2 μl, 5 μl, and 10 μl, respectively, of 50ng/μl λHindIII ladder. Lastly, load 5 μl per lane of DNA extract with dye for each sample. </li>
 <li>Run the gel at 15 volts for approximately 16 hours (we recommend running overnight). </li>
 <li>The next day, photograph the gel using a UV gel documentation system. To determine the molecular weight range of your DNA and its concentration, compare the sample bands to bands of the ladders. Good quality DNA will have high molecular weight (&gt;36kb), and show little evidence of shearing/degradation. See Figure 1.</li>
</ol>
Part Five: Cesium Chloride Gradient Centrifugation
<ol>
 <li>If the DNA quality is good and quantity is sufficient, you can proceed to purify the DNA by performing a cesium chloride (CsCl) gradient centrifugation. Note that this purification step is optional and is not necessary for all downstream applications (for example, if you want to generate fosmid libraries you SHOULD purify, whereas if you simply want to perform PCR reactions, purification is not usually necessary). First, label one centrifuge tube for each sample. </li>
 <li>To each centrifuge tube, add 160 mg of CsCl, 178 μl of genomic DNA. After adding CsCl and DNA to the tube, place a small piece of parafilm on top of the tube and gently invert the tube ten to twenty times in order to mix the components. Do not mix by pipetting, which may cause shearing of the DNA. Next, add 10 μl of 10 μg/μl EtBr to this tube and mix it in the same way. (Be extremely cautious when using EtBr- it is a known mutagen and suspected carcinogen. Make sure to change gloves often and avoid EtBr transfer to the rest of the lab and to your skin.)</li>
 <li>Make sure that weight of all the tubes are balanced before centrifugation such that the weight differences among the tubes are less than 1 mg. </li>
 <li>Place the tubes in the rotor using hemostat, close the lid and place the rotor inside the ultracentrifuge. </li>
 <li>Close the ultracentrifuge door. Vacuum will be applied as soon as you close the door. Run overnight at 100,000 rpm for 18 hours and 20°C. </li>
 <li>When centrifugation is complete, take out the tubes from the rotor using hemostat and place on a tube rack. </li>
 <li>Visualize the DNA with blue light transilluminator, or if unavailable, use long wavelength UV light, in a dark room. Wear a pair of amber filter glasses to see the DNA band. See Figure 2. </li>
 <li>Using a sterile 1cc syringe and needle (26G 5/8), remove DNA band and place it in a 1.5ml Eppendorf tube. </li>
 <li>To help recover most of the DNA trapped in the dead space in the syringe, rinse syringe with 100 μl TE and add the rinsed solution to the tube. Prepare one tube with 100 μl TE buffer per sample in order to prevent cross contamination. </li>
 <li>To remove EtBr from the DNA, add an equal volume of water-saturated butanol to the tube, invert the tubes gently ten to twenty times, centrifuge at 10000 rpm for 1 minute and discard the top layer. </li>
 <li>Repeat washing until the color of butanol is transparent, 3 - 4 more times. </li>
 <li>Place 4 ml TE in an Amicon Ultra centrifuge tube and add the DNA solution from above. </li>
 <li>Centrifuge at 3500 g at room temperature, until the DNA volume is reduced to approximately 100-500 μl (approximately 6-7 minutes) and discard the flow-through. </li>
 <li>Add 2ml TE to Amicon filter and centrifuge at 3500 g for 6 minutes, making sure to use a fresh pipette tip for each addition to avoid cross contamination. Repeat twice. </li>
 <li>Concentrate to a final volume of 50-100 μl by additional centrifugation as necessary and transfer the DNA solution on the filter to a new 1.5 ml Eppendorf tube. </li>
 <li>Add 40 μl TE to the Amicon filter and pipette up and down along both filter membranes to wash out any remaining DNA. Add this solution to the tube in 5.15. </li>
 <li>Place a Microcon YM-30 filter unit into a Microcon tube and prewash the Microcon by adding 200 μl autoclaved water and centrifuging at 10000 rpm for 7 minutes. </li>
 <li>Add the DNA solution from 5.16 to the prewashed Microcon in order to further concentrate the DNA. Centrifuge at 5000-10000 g for 1 - 3 minutes. Check the amount of liquid on the filter and repeat centrifugation until the amount of liquid on the filter is reduced to approximately 50 μl.</li>
 <li>Place the filter unit upside down in a new Microcon tube and centrifuge at 1000 g for 3 minutes. The ideal amount of concentrated solution is 50-60 μl. </li>
 <li>Measure and record the concentration of the DNA on a Nanodrop and check peak quality (should be 260nm).</li>
 <li>Transfer a small aliquot of the DNA to a clean eppendorf tube for working stock and freeze at minus 20. Transfer the rest of the DNA to a second clean eppendorf and freeze at minus 80.</li>
</ol>
Representative Results:
When this protocol is done correctly, you should see a gel image after step 4.4 in Determination of DNA quality similar to Figure 1. Actual DNA concentration of extracts will vary depending on the source of the sample. After step 5.7 in CsCl gradient centrifugation, genomic DNA illuminated by blue light should look similar to Figure 2.
<img src="/files/ftp_upload/1352/1352Fig1newer.png" alt="Gel electrophoresis diagram: DNA separation, molecular weight markers visible, showing band migration." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. 0.8% agarose gel electrophoresis image of high molecular weight DNA collected from four depths in the subarctic Pacific Ocean (in duplicate), stained with the intercalating agent ethidium bromide (10 mg/ml). Gel was run at 15V for ~16hrs in 1X TBE gel running buffer. Sample bands are of good quality showing little evidence of mechanical shearing (shows single bands or smears as opposed to multiple bands) although the 10m extracts retain some RNA carry over (see smear in the 0.5 to 2.0 Kb range). 
<img src="/files/ftp_upload/1352/1352Fig2.jpg" alt="Fluorescence in test tubes; chromatography process for DNA separation using UV light." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2.Genomic DNA band illuminated by blue light after CsCl gradient centrifugation.

<ol><li>Beja, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((To+BAC+or+not+to+BAC%3A+marine+ecogenomics%5BTitle%5D)+AND+%22Curr+Opin+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Construction+and+analysis+of+bacterial+artificial+chromosome+libraries+from+a+marine+microbial+assemblage%5BTitle%5D)+AND+%22Environ+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Community+genomics+among+stratified+microbial+assemblages+in+the+oceans+interior%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+uncultivated+prokaryotes%3A+isolation+and+analysis+of+a+40-kilobase-pair+genome+fragment+from+a+planktonic+marine+archaeon%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">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/pubmed?term=((A+simple%2C+rapid+method+for+the+direct+isolation+of+nucleic+acids+from+aquatic+environments%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">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

<table><tbody><tr><td>1 cc syringe (26G5/8 needle)</td><td>BD Biosciences</td><td>309597</td><td></td></tr><tr><td>1 ml syringe (26G5/8 needle)</td><td>BD Biosciences</td><td>309597</td><td></td></tr><tr><td>1.5ml eppendorf tubes</td><td>Eppendorf</td><td>0030 125.150</td><td></td></tr><tr><td>15ml tubes</td><td>Sigma-Aldrich</td><td>CLS430791</td><td></td></tr><tr><td>5cc syringe</td><td>BD Biosciences</td><td>309603</td><td></td></tr><tr><td>Amicon&amp;#174; Ultra-4 Centrifugal Filter Devices</td><td>EMD Millipore</td><td>UFC801096</td><td>10,000 Nominal molecular weight limit</td></tr><tr><td>Butanol</td><td>Fisher Scientific</td><td>A383-1</td><td>Make it water saturated (butanol:water = 1:1)</td></tr><tr><td>Centrifuge rotor, JA5.3 </td><td>Beckman Coulter Inc.</td><td>368690</td><td></td></tr><tr><td>Centrifuge, Avanti&amp;#174; J-E</td><td>Beckman Coulter Inc.</td><td>369003</td><td></td></tr><tr><td>Cesium chloride</td><td>Sigma-Aldrich</td><td>C4036</td><td></td></tr><tr><td>Chloroform:IAA (24:1)</td><td>Sigma-Aldrich</td><td>25666-500ML</td><td></td></tr><tr><td>Ethidium Bromide (EtBr)</td><td>Sigma-Aldrich</td><td>E1510-10ml</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Hybridization oven</td><td>Fisher Scientific</td><td>13-247-10</td><td>37&amp;#x00B0;C &amp;amp; 55&amp;#x00B0;C</td></tr><tr><td>Lysis Buffer</td><td></td><td></td><td>0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3)</td></tr><tr><td>Lysozyme</td><td>Sigma-Aldrich</td><td>L6876-5G</td><td>125 mg in 1000 &amp;#x03BC;l TE</td></tr><tr><td>Microcon&amp;#174; Ultracel YM-30 Centrifugal Filter Unit</td><td>EMD Millipore</td><td>42410</td><td>50 bp cut off</td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-10</td><td></td></tr><tr><td>Phenol:Chloroform:IAA (25:24:1)</td><td>Sigma-Aldrich</td><td>77617-500ML</td><td></td></tr><tr><td>Proteinase K</td><td>Qiagen</td><td>19133</td><td></td></tr><tr><td>RNase A</td><td>Fisher Scientific</td><td>BP2539-100</td><td>10 &amp;#x03BC;g/ml</td></tr><tr><td>Safe Imager&amp;#8482; Blue light transilluminator</td><td>Invitrogen</td><td>S37102</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma-Aldrich</td><td>L4509</td><td>20% SDS</td></tr><tr><td>Sterivex filters</td><td>Fisher Scientific</td><td>SVG010RS</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Table top centrifuge</td><td>Eppendorf</td><td>5415D</td><td></td></tr><tr><td>TE buffer, pH 8.0</td><td></td><td></td><td></td></tr><tr><td>Trizma&amp;#174; base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Ultracentrifuge rotor, TLA 100</td><td>Beckman Coulter Inc.</td><td>343847</td><td></td></tr><tr><td>Ultracentrifuge tube (7X20mm, PC)</td><td>Beckman Coulter Inc.</td><td>343775</td><td></td></tr><tr><td>Ultracentrifuge tube rack</td><td>Beckman Coulter Inc.</td><td>348302</td><td></td></tr><tr><td>Ultracentrifuge, Optima&amp;#174; Max</td><td>Beckman Coulter Inc.</td><td></td><td></td></tr></tbody></table>

dna extraction from 0.22 &mu;m sterivex filters and cesium chloride density gradient centrifugation

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 ...

Hi, I'm Jody from the laboratory, Steven Hallam in the Department of Microbiology in immunology at the University of British Columbia. Today we'll show you a procedure for extracting genomic DNA, concentrated from seawater samples onto 0.2 micron STX filters and a purification of this DNA by a cesium chloride density gradient centrifugation. We use this procedure in our laboratory to obtain environmental DNA so that we can study microbial community structure, diversity and metabolism in marine environments.So let's get started. Begin this protocol by thawing on ice, the stir of X filters that you have previously concentrated with planktonic biomass, treated with lysis, buffer and frozen for simplicity. Here we describe the procedure for processing an individual filter.In practice we recommend processing 15 or fewer filters at a time. Once the filter has thawed, begin the first of two incubations. One to lyce the cells and remove RNA and one to break down proteins.For the first incubation, add 100 microliters lysozyme, and 20 microliters RN a's a to the filter. Reseal the filter with parfum and leave it to rotate at 37 degrees Celsius for one hour. For the next incubation, add 100 microliters protein ace KN 100 microliters 20%SDS to the filter.Reseal the filter using perfil and leave it to rotate one to two hours more. This time at 55 degrees. Use a five cc syringe to transfer the lysate from the Stax filter to a 15 milliliter falcon tube.Rinse the filter with one milliliter lysis buffer and add the rinse liquid to the lysate in your falcon tube. Now that you have lysed and digested your cells, you are ready to extract their DNA. The next step is to use the phenol chloroform method to extract the DNA from your lysate.Add an equal volume about three milliliters of phenol chloroform, isoamyl alcohol to the lysate tube vortex. For 10 seconds to mix well spin the tube at 2, 500 times G for five minutes. During centrifugation, the phenol chloroform lysate mixture should separate into two layers.An organic layer on the bottom containing the phenol chloroform and proteins from your sample. And an aqueous layer usually on top, which consists of your DNA water and other more hydrophilic molecules. Transfer the aqueous layer containing your DNA into a new 15 milliliter Falcon tube To this tube, add an equal volume of a chloroform isoamyl alcohol mixture vortex for 10 seconds.This step removes any remaining phenol from your DNA sample. Spin it 2, 500 times G for five minutes or until the aqueous layer is clear. Transfer the aqueous layer into a new labeled falcon tube and add one milliliters of TE at PH eight.Now you have your DNA extract. The next step is to wash and concentrate the DNA sample in a 15 milliliter Amazon ultracentrifuge tube. Transfer your DNA sample to the filter compartment of anon.Ultra tube theon tubes consists of a filter that retains molecules at or above a specified molecular weight, the tate and a tube to catch the flow through the filtrate. In this case, the filter retains your DNA spin at 3, 500 times G for 10 minutes. Check to make sure there is less than one milliliter of liquid retained in the Amon filter.If more reten Tate remains, refill the filter with TE and spin again. Remove the flow through or filtrate to another falcon tube and save it in the fridge until you have confirmed your final product on a gel, add two milliliters TE buffer to the Amazon filter and spin at 3, 500 times G for six minutes. Remove the filtrate as before.Wash the DNA twice more with TE for a total of three washes, saving the filtrate each time for the last wash spin until 200 to 500 microliters of Tate remain in the Amon filter. Note the final volume and transfer to a labeled 1.5 milliliter einor tube. Make a 70 microliter aliquot of the washed concentrated DNA to use as working stock.Store the working stock at minus 20 degrees Celsius. Freeze away the rest of the DNA at minus 80 degrees Celsius. Check the DNA concentration and quality of your final product by running out your samples on a gel next to DNA.Ladders which serve as size and intensity standards set up a 0.8%AROS gel plus atherium bromide to run overnight. In the first few lanes load ladders with bands of various molecular weights and concentrations, we recommend the following loading pattern. In the first two lanes, load 10 microliters of one kilobyte plus ladder in the next three lanes, load two microliters, five microliters, and 10 microliters respectively of 50 nanograms per microliter Lambda H three ladder.Lastly, load five microliters per lane of DNA extract sample. Run the gel at 15 volts for approximately 16 hours. Photograph the gel using the UV gel documentation system to determine the molecular weight range of your DNA and its concentration.Compare the sample bands to bands of the ladder. Good quality DNA will have high molecular weight and show little evidence of shearing or degradation. If the DNA quality is good and quantity is sufficient, proceed to purify the DNA by performing a cesium chloride gradient.Centrifugation first label, one centrifuge tube for each sample to each centrifuge tube, add 160 milligrams of cesium chloride and 178 microliters of genomic DNA. Cover the tube with a small piece of perfil and gently invert the tube 10 to 20 times in order to mix the components. Do not mix by pipetting which may cause shearing of the DNA.Then add 10 microliters of AUM bromide to the tube and mix again. Make sure that the tubes are balanced well before centrifugation and that the weight differences among the tubes are less than one milligram. Put the tubes into the ultracentrifuge rotor using tweezer scissors.Then close the rotor lid and place the rotor inside the ultracentrifuge close. The ultracentrifuge door vacuum will be applied as soon as you close the door. Run overnight for a total of 18 hours at 100, 000 RPM at 20 degrees Celsius.When centrifugation is complete, take out the tubes from the rotor using tweezer scissors and place on the rack. Take the tubes over to the blue light transluminator, put on the amber filter glasses. Then turn on the blue light and behold your DNA band using a sterile one cc syringe n needle.Remove the DNA band and then place it in a 1.5 milliliter einor tube to help recover most of the DNA trapped in the dead space in the syringe. Rinse the syringe with 100 microliters te and add the rinse solution to the tube. Prepare one tube with 100 microliters te buffer per sample in order to prevent cross-contamination.To remove AUM bromide from the DNA, add an equal volume of water saturated butanol to the tube. Invert the tubes gently, 10 to 20 times centrifuge at 10, 000 RPM for one minute and discard the top layer. Repeat washing about three to four more times until the color of the butanol is transparent.Place four milliliters of TE in ancon ultracentrifuge tube and add the DNA solution from above centrifuge at 3, 500 times G at room temperature until the DNA volume is reduced to approximately 100 to 500 microliters and discard the flow through. Add two milliliters TE to the Amazon filter and centrifuge at 3, 500 times G for six minutes. Repeat twice for a total of three washes with te concentrate to a final volume of 50 to 100 microliters by additional centrifugation as necessary.And transfer the DNA solution on the filter to a new 1.5 milliliter einor tube. Add 40 microliters te to theon filter and pipette up and down along both filter membranes to wash out any remaining DNA. Add this solution to the tube of concentrated DNA.Place a MicroCon YM 30 filter unit to a MicroCon tube and pre-wash the MicroCon by adding 200 microliters autoclave water and centrifuging at 10, 000 RPM for seven minutes. Add the DNA solution to the pre-washed MicroCon in order to further concentrate the DNA centrifuge at 5, 000 to 10, 000 times G for one to three minutes. Check the amount of liquid on the filter and repeat centrifugation until the amount of liquid on the filter is reduced to approximately 50 microliters.Place the filter unit upside down in a new MicroCon tube and centrifuge at 1000 times G for three minutes. The ideal amount of concentrated solution is 50 to 60 microliters. Transfer your DNA sample to a new 1.5 milliliter einor tube.Measure the concentration of DNA on a NanoDrop and check the peak quality. A graph of absorbances for clean DNA should have a clear peak at 260 nanometers. Freeze the rest of the DNA at negative 80 degrees Celsius.When this protocol is done correctly, you should see a gel image similar to figure one actual DNA concentration of extracts will vary depending on the source of the sample. When this protocol is done correctly, you should clearly see a DNA band across the middle of the tube. As in figure two, We've just shown you how to extract genomic DNA from sterex filters and purify it using a caesium chloride density gradient.When doing this procedure, it's important to check before you begin that you have all the necessary reagents and expendable items. And secondly, plan at least three full days to allow plenty of time to complete the procedure carefully. So that's it.Thanks for watching and good luck with your experiments.

dna-extraction-from-022-m-sterivex-filters-cesium-chloride-density

Research

JoVE Journal

Biology

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Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Learn DNA extraction from Sterivex filters and cesium chloride purification for environmental DNA studies. This method enables high-quality genomic DNA recovery from marine planktonic biomass.

DNA Extraction from 0.22 μM Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

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