Name: Extraction of High Molecular Weight Genomic DNA from Soils and Sediments (Video) | JoVE Journal
Uploaded: 2009-11-10T20:30:00.000+00:00
Duration: 11 min 24 s
Description: Learn high molecular weight genomic DNA extraction from soil and sediment samples for microbial community analysis. This method enables construction of metagenomic libraries to study diversity and metabolic potential.

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We would like to thank the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund, Genome British Columbia and the British Columbia Ministry of Forests and Range for supporting ongoing studies of forest soil productivity. S.L was supported by a fellowship from the TULA foundation funded Centre for Microbial Diversity and Evolution.

Part I: Extraction of DNA
Procedure
1. 8 hours are required, for processing 6 samples excluding quantification of DNA.
Before you start the extraction, you will need to pre-chill mortar, pestle and spatula in a -20 °C freezer or using liquid nitrogen. Also, pre-chill 50 ml tubes containing 20 ml chloroform isoamyl alcohol (24:1) on ice. Set the hybridization oven to 65 °C to pre-heat. Check CTAB solution, if it is crystallized warm to 60 °C to melt the crystals. Complete the denaturing solution and extraction buffer by adding the last ingredients just before you start the extraction (see the recipe below). Conduct all extraction procedures in a fume hood.
2. Prepare the denaturing buffer and extraction buffer. Note: To maximize the DNA yield, it is critical to precisely make buffers following the instructions. 
3. Place 2 g of soil in a pre-chilled mortar. Note: Soil collected in the field should be frozen on dry ice for transport and stored at -80 °C. Thaw and sieve soil using a 2 mm sieve prior to DNA extraction. You can increase the starting amount of soil up to 10 g.
4. Add 1 ml of denaturing solution to the soil.
5. Freeze and grind the soil in liquid N2 using a pestle until the soil particles look powdery and homogenous. Repeat freezing and grinding twice. Note: Keep the soil sample frozen during the grinding, using enough liquid nitrogen to fully cover the soil sample. Slight melting during the grinding is acceptable.
6. Transfer the ground soil to a 50 ml conical tube using a pre-chilled spatula. Note: The ground soil sample can be stored at -80 °C at this point, if necessary. 
7. Add 9 ml of extraction buffer. To mix the solution and soil, vortex briefly at low speed. Note: To ensure the buffer is homogeneous, warm it at 60 °C for 10-15 minutes before use. Keep the rest of buffer at 60 °C throughout the extraction. Vortex at speed 8 (on a scale where 10 is the maximum). 
8. Incubate for 40 min at 65°C in a hybridization oven. During the incubation, continuously rotate the tubes at the lowest speed, or gently invert the tubes every 10 min. Note: Place the tube horizontally in a hybridization oven to allow buffer to contact soil over a large surface area. The solution may look a bit viscous during incubation.
9. Centrifuge at 1800 xg, for 10 min at 10 - 14 °C. Transfer supernatant into the pre-chilled 50 ml conical tube containing 20 ml chloroform-isoamyl alcohol (24:1), keep on ice. Note: When you transfer supernatant, be careful not to disturb the organic layer (bottom layer). Leave 1-2 ml of supernatant if it is necessary in order to collect only clean supernatant. You often can see a beige layer (approximately 1-3 mm in thickness) on top of the supernatant. Do not disturb this layer when collecting the supernatant. Try to slide the tip along the tube wall to prevent breakage of this beige layer. If the supernatant is too turbid, repeat the chloroform-isoamyl alcohol extraction one more time on the supernatant. Do not use disposable plastic pipettes when transferring chloroform, as chloroform will melt the plastic. Use a glass pipette, or a glass graduated cylinder, or pour chloroform directly into 50 ml conical tubes.
10. Extract the soil pellets 2 more times;
10.1. Add 5 ml extraction buffer to the soil pellets, gently mix using 1 ml tip followed by brief vortexing at low speed. Note: Shake and mix the buffer before use. 
10.2. Incubate at 65°C for 10 min in hybridization oven. Centrifuge at 1800 xg, for 10 min at 10 - 14 °C. Transfer supernatant to the tube at step 9. Keep the tube with collected supernatant and chloroform-isoamyl alcohol on ice during the extraction. Note: Make sure you add the supernatant to the same tube without cross mixing between the samples.
10.3. Repeat 9.1 and 9.2 one more time.
11. Using a rotating plate, very gently rock the tube that contains the collected supernatant (14-19 ml) and chloroform-isoamyl alcohol at 1.25 rpm for 10 min. Note: Close the lid very tightly to prevent leakage. Lay a paper towel on the rocking plate before you place the tube so any leaks can be readily detected. 
12. Centrifuge at 1800 xg, for 20 min at 10 - 14 °C.
13. Transfer aqueous phase to a new tube. Note: Do not disturb the interphase between the aqueous phase (top layer) and organic layer (bottom layer) when you transfer the supernatant. Leave approximately 1.5- 2 ml supernatant to ensure collection of only clean supernatant.
14. Recover the DNA using isopropyl alcohol (step 15.a - 21.a). Alternatively, use Amicon® Ultra-15 Centrifugal Filter Devices (step 15.b - 21.b) if you expect very low amount of biomass in your sample, which will produce hardly visible pellet by isopropyl alcohol precipitation that is difficult to recover.
15.a. Place the supernatant to a new tube ultracentrifuge.
16.a. Add isopropyl alcohol to the collected supernatant at a volume ratio of 0.6:1. Invert the tubes gently a few times to mix.
17.a Incubate for 30 min at room temperature.
18.a. Centrifuge at 16000 xg, for 20 min at 20 - 25 °C. Ensure the weights of all the tubes are balanced well before the centrifugation step. Note: Mark the direction of centrifugal force on the tube, so you can detect the DNA pellet more easily when the DNA yield is low.
19.a. Remove isopropyl alcohol by decanting or vacuum suctioning. Be careful not to disturb the DNA pellet. Note: Remove isopropyl alcohol as soon as centrifugation is completed. Be very careful not to lose the DNA pellet. The size of DNA pellet can vary widely depending on the soil type, from a clearly visible (~9 mm diameter) to hardly visible. It is recommended to remove the residue of isopropyl alcohol by suctioning along the wall of the tube carefully so as not to suction out the DNA pellet. 
20.a. Dry DNA pellet at room temperature for 5 - 20 min. Note: Do not over dry DNA pellet, otherwise it will be difficult to redissolve the DNA.
21.a. Resuspend the DNA pellet in 200 μl TE. Mix it by gentle tapping. Transfer the DNA solution into 1.5 ml tube. Keep the tube at 4°C overnight in order to fully dissolve DNA. Note: Depending on the size of pellet, you can adjust the volume of TE to add. Usually 200 - 400 μl of TE works well.
*Alternatively follow the steps 15.b - 21.b.
15.b. Pre-wash an Amicon® Ultra-15 Centrifugal Filter Devices; add 15 ml TE to the device and centrifuge at 3500 xg, for 10 min. Discard the flow-through.
16.b. Place the DNA solution from step 13 to the pre-washed Amicon filter. Centrifuge at 3500 xg until the DNA volume is reduced to 200 - 500 μl. Discard the flow-through.
17.b. Wash the DNA with TE twice; add 10 ml TE to the Amicon filter devices. Centrifuge at 3500 xg until the DNA volume is reduced to approximately 200 μl. Discard the flow-through. Repeat one more time.
18.b. Transfer the DNA solution on the filter to a new 1.5 ml tube. 
19.b. Add 40 μl TE to the Amicon filter. Wash both side of filter membranes by pipetting up and down in order to recover any DNA residue on the filter. Add this wash solution to the tube at step 18.b.
20.b. If necessary, the DNA can be further concentrated by Microcon® Ultracel YM-30 Centrifugal Filter Unit; pre-wash the filter by adding 200 μl TE and centrifuging at 10000 xg for 7 minutes. Add the DNA solution to the filter and centrifuge at 5000 xg for 1 min. Repeat centrifugation until the amount of DNA solution on the filter reduced to 50 μl approximately. Note: Maximum initial sample volume for Microcon filter is 500 μl. Do not centrifuge at speeds over 14000 x g. Do not over centrifuge until the membrane dries completely. Ensure that some liquid still covers the filter after centrifugation. If you over centrifuged and the membrane is dry, then add 15 μl TE, agitate gently for 30 seconds, and proceed to step 21.b.
21.b. Place the filter unit upside down in a new Microcon tube and centrifuge at 1000 x g for 3 min to recover the DNA. 
22. Quantify DNA by TAE gel (0.8%, 0.5xTAE, 50 V for 4 hours), and by Nanodrop spectrophotometer or Pico-green assay and plate reader. Note: DNA amount can be estimated by comparing the sample band to the marker band with known concentration (High molecular DNA marker or λHindIII) on the gel. 
23. Check the integrity of high molecular weight DNA using Pulsed Field Gel Electrophoresis (PFGE) with 1%, 1xTAE gel (For more details see the Step II, part2 at <a href="https://www.jove.com/index/details.stp?id=1387">https://www.jove.com/index/details.stp?id=1387</a>, doi: 10.3791/1387 by Taupp et al. 2009). Note: You can use regular agarose gel for this QC purpose instead of low melting agarose. In brief, electrophoresis conditions are as follows: 2-250 kb, 6 volts, initial switching time: 5 sec, final switching time: 15 sec, run for 12 hrs at 14 °C. SYBR Gold staining for one hour to visualize the DNA on the gel. 
24. If the molecular weights of extracted DNA are satisfactory, then proceed to the following CsCl gradient centrifuge step. Alternatively, DNA can be stored at -20 °C or -80 °C before the CsCl centrifuge. The median size of the DNA should be the equal or exceed 36 Kb, if you want to construct a large insert metagenomic DNA library. 
Part II. DNA purification using cesium chloride gradient ultracentrifugation
Preparation of centrifuge: 1.5 - 2 hours Centrifugation step: 18 hours Removing ethidium bromide (EtBr) and concentrating DNA after centrifugation: 3 hours
Procedure
For details, refer Wright et al. 2009. <a href="https://www.jove.com/index/details.stp?id=1352">https://www.jove.com/index/details.stp?id=1352</a>, doi: 10.3791/1352
Representative Results/Outcome
The total amount of genomic DNA isolated from 2 g of forest soil organic (O horizon) and mineral horizons (Ae, AB, and Bt horizons) (sampled from the Long Term Soil Productivity (LTSP) site located at Skulow lake, BC, Canada and managed by BC Ministry of Forests and Range) ranged from 10 mg to 180 mg. The median size of isolated DNA was usually between 40 - 60 kb (Figure 1). The ratio of absorbance at 260 nm/280 nm was between 1.3-1.6 and there was a peak at 230 nm, which may indicate humic acid contamination often observed in soil samples. The CsCl gradient centrifugation drastically reduced this contamination and also increased the ratio of 260/280 to 1.8 - 1.9 as shown in Figure 2a and 2b.
<img src="/files/ftp_upload/1569/1569fig1.jpg" alt="DNA separation gel electrophoresis result; diagram; bands indicating size and quantity of DNA fragments." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. PFGE pictures of the extracted genomic DNA before CsCl ultracentrifugation. lane1: 1 kb marker 100 ng, lane 2: λHind III marker 100 ng, lane 3: λHind III marker 250 ng, lane 4: λHind III marker 500 ng, lane5: Fosmid 36 kb marker 100 ng, lane 6 and 7: genomic DNA isolated from the mineral soil horizon Ae, lane 8 and 9: genomic DNA isolated from the mineral soil horizon AB at the LTSP site located Skulow lake, BC 
<img src="/files/ftp_upload/1569/1569fig2tmb.jpg" alt="Spectroscopy graphs, absorption spectra, UV-Vis, diagram, wavelength analysis, scientific data." border="0" data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2. Chromograms of genomic DNA detected by spectrophotometer before (A) and after CsCl ultracentrifugation (B). Note the reduction in the absorbance value at the 230 nm wavelength after CsCl ultracentrifugation, which indicates improvement in the genomic DNA quality. Please <a href="https://www.jove.com/files/ftp_upload/1569/1569fig2.jpg">click here</a> to see a larger version of figure 2.

<ol><li>Hurt, R. A., Qiu, X., Wu, L., Roh, Y., Palumbo, A. V., Tiedje, J. M., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+recovery+of+RNA+and+DNA+from+soils+and+sediments%5BTitle%5D)+AND+%22AEM%22%5BJournal%5D)">Simultaneous recovery of RNA and DNA from soils and sediments.</a> AEM. 67 (10), 4495-4503 (2001).</li>
<li>Wright, J. J., Lee, S., Zaikova, E., Walsh, D. A., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+extraction+from+0.22+%CE%BCM+Sterivex+filters+and+cesium+chloride+density+gradient+centrifugation%5BTitle%5D)+AND+%22JoVE%22%5BJournal%5D)">DNA extraction from 0.22 μM Sterivex filters and cesium chloride density gradient centrifugation.</a> JoVE. , (2009).</li>
<li>Taupp, M., Lee, S., Hawley, A., Yang, J., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large+insert+environmental+genomic+library+production%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Large insert environmental genomic library production.</a> J Vis Exp. , (2009).</li>
</ol>

<table><tbody><tr><td colspan="4">&lt;strong&gt;*Denaturing solution&lt;/strong&gt;: 10 ml in total</td></tr><tr><td>Guanidine isothiocyanate (MW 118.16), 4.73 g</td><td></td><td></td><td></td></tr><tr><td>1M Tris-HCl (pH 7.0), 100 &amp;mu;l</td><td></td><td></td><td></td></tr><tr><td>0.5M EDTA, 20 &amp;mu;l</td><td></td><td></td><td></td></tr><tr><td colspan="4">Add water to 9.95 ml in total.</td></tr><tr><td colspan="4">Autoclave.</td></tr><tr><td colspan="4">Add 50 &amp;#x03BC;l 2-mercapt&amp;#8211;thanol just before use. (5 &amp;#x03BC;l of 2-mercapt&amp;#8211;thanol per 1 ml Denaturing Solution)</td></tr><tr><td colspan="4">Note: Keep the Denaturing solution at 4 &amp;deg;C. Do not use buffer older than one week. If possible, make fresh buffer to use.</td></tr><tr><td colspan="4">&lt;strong&gt;**Extraction Buffer&lt;/strong&gt;</td></tr><tr><td>1M Sodium phosphate buffer [pH 7.0]*, 100 ml</td><td></td><td></td><td></td></tr><tr><td>1M Tris-HCl [pH 7.0], 100 ml</td><td></td><td></td><td></td></tr><tr><td>0.5M EDTA [pH 8.0], 200 ml</td><td></td><td></td><td></td></tr><tr><td>5 M NaCl, 300 ml</td><td></td><td></td><td></td></tr><tr><td colspan="4">Autoclave and keep it at room temperature.</td></tr><tr><td colspan="4">Add 200ml, 5% Hexadecyltrimethylammonium bromide (CTAB, autoclaved) and 100 ml, 20% SDS (autoclaved) just before use. If CTAB was crystallized, melt it at 60 &amp;deg;C.</td></tr><tr><td colspan="4">* 1M Sodium phosphate buffer: 57.7 ml, 1M Sodium phosphate monobasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;) and 42.3 ml, 1M Sodium phosphate dibasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;). Adjust pH to 7.0</td></tr><tr><td colspan="4">Note: Do not use 20 % SDS if it has precipitation. It is normal to see milky suspension when you add SDS to the solution. Once you add SDS, place the extraction buffer at 60 &amp;deg;C to ensure SDS is well suspended.</td></tr></tbody></table>

extraction of high molecular weight genomic dna from soils and sediments

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 ...

Hi, I am San Lee from the laboratory of Stephen Harlem in the Department of Microbiology and Immunology at the University of British Columbia. Today we show you a procedure for high molecular weight genomic, DNA extraction from forest to soil that is also applicable to a wide variety of soil and sediment types. We use high molecular weight genomic, DNA to construct the forest meter library of soil microbial communities.With these libraries in hand, we can study the diversity and metabolic potential of microbial communities partitioning among different soil horizons. So let's get started. This protocol begins with cell lysis.The first step is to complete the denaturing buffer by adding betta mer capto ethanol to a concentration of 0.5%For best results, make the denaturing solution fresh each time. Alternatively, use buffer less than one week old that has been kept at four degrees Celsius. For the next step, you'll need a doer of liquid nitrogen and an autoclave and pre chilled mortar pestle and spatula.Place a two gram frozen soil sample in the pre chilled mortar. Add one milliliter of the denaturing solution. Then add liquid nitrogen to fully cover the soil.Use a pre chilled pestle to grind the sample until the soil particles look powdery and homogenous and the sample begins to melt. Then add more liquid nitrogen and repeat the grinding step. This completes the cell lysis step using a pre chilled spatula.Transfer the ground sample to a 50 milliliter conical tube. Keep the sample on ice to proceed immediately to the DNA extraction step or store at minus 80 degrees Celsius. For liter use, just before beginning the DNA extraction, complete the extraction buffer by adding hexa deco trimethyl ammonium bromide or CTAB and SDS.First.Check if the CTAB is crystallized, and if so, dissolve it at 60 degrees Celsius before proceeding. Then add it to the solution for a total concentration of 1%Next, add 20%SDS solution to a final concentration of 2%The milky suspension. That forms is normal.Once the SDS has been added, keep the completed extraction buffer homogenous by storing it at 60 degrees Celsius after 10 to 15 minutes. Once the extraction buffer is well dissolved and homogenous, add nine milliliters of extraction buffer to the soil sample and vortex briefly at low speed to mix. Incubate the sample with extraction buffer in a 65 degree Celsius hybridization oven for 40 minutes.During the incubation, gently invert the tubes every 10 minutes or continuously rotate the tubes at the rotor's lowest speed. During incubation, the solution may appear slightly viscous after the incubation. The next step is to use chloroform isoamyl alcohol to purify the DNA away from the organic material in the lysis mixture.Begin by centrifuging the sample for 10 minutes at 1800 Gs at about 10 to 14 degrees Celsius in the hood, collect the DNA containing supernatant by sliding your pipette tip carefully along the side of the tube, avoiding the beige layer on top and the organic layer on the bottom. Transfer the supernatant to a pre chilled 50 milliliter tube containing 20 milliliters of chloroform ISL alcohol or i a a next, repeat the extraction process two more times on the same sample. Meanwhile, keep the DNA extract and chloroform i a a on ice in the hood.For the second DNA extraction, go back to the tube containing the soil pellet with residual extraction buffer and add five more milliliters of extraction buffer. Stir gently with a one milliliter tip to mix and vortex briefly to resuspend the pellet as before. Incubate at 65 degrees Celsius this time for 10 minutes.Then centrifuge at 1800 Gs for 10 minutes. At about 10 to 14 degrees Celsius in the hood, transfer the supernatant to the tube containing chloroform, isoamyl alcohol or chloroform i a a and the supernatant from the previous extraction. Repeat the entire extraction process one more time to extract as much DNA as possible from the soil sample.When you have completed a total of three extractions on your soil sample, very gently shake the tube containing the collected supra natant and chloroform i a a on a rotating plate at 1.25 RRP m for 10 minutes After this mixing step, centrifuge the tube at 1800 Gs for 20 minutes at about 10 to 14 degrees Celsius. After centrifugation, carefully transfer the aqueous phase to a new ultra centrifuge tube, leaving one to two milliliters of SNAT in order to avoid disturbing the interface between the aqueous and organic phases. Now to precipitate the DNA add 0.6 milliliters of isopropyl alcohol.To each milliliter of collected supernatant, invert the tubes gently a few times to mix. At this point, you may be able to see the DNA precipitate into long threads. Then incubate the DNA with the isopropanol for 30 minutes at room temperature.After the incubation, it is time to pellet the DNA by centrifugation mark one corner of the tube as you place it into the centrifuge so that you know where to expect the DNA pellet Spin at 16, 000 GS for 20 minutes at 20 to 25 degrees Celsius following centrifugation, remove the isopropyl alcohol by decanting and or vacuum suctioning. Be careful not to lose the DNA pellet, which ranges in size from hardly visible to nine millimeters wide, depending on extraction efficiency and biomass in soil samples, dry the DNA pellet at room temperature for five to 20 minutes. Careful not to over dry when the pellet is dry.Resuspend the DNA in 200 to 400 microliters of te. Mix it by gentle tapping. Transfer the DNA solution into a 1.5 milliliter tube.Keep the DNA at four degrees Celsius overnight. In order to fully dissolve the DNA once the DNA has been extracted from the soil sample, the remaining steps for checking the concentration and DNA integrity of the sample are standard procedures, which are described in detail in another JoVE protocol from the Hallam laboratory. Determine the DNA concentration using the method of your choice.Also check the integrity of high molecular weight DNA by running out 10 to 20 microliters of your DNA sample using pulsed field gel electrophoresis or PFGE. The median DNA size should be 36 kilobases or greater depending on your downstream application. Proceed to the following cesium chloride gradient centrifuge step alternatively, DNA can be stored at minus 20 degrees Celsius or minus 80 degrees Celsius before ultracentrifugation.The next step is use cesium chloride gradient centrifugation to further purify the DNA by removing impurities, following cesium chloride gradient centrifugation concentrate, and further purify the DNA using AmCon and MicroCon centrifugal filter devices. The cesium chloride gradient centrifugation is key to achieving high quality DNA and is described in detail in another JoVE protocol from the Hallam laboratory. After performing the cesium chloride gradient centrifugation and concentration steps, check the DNA concentration using the method of your choice.Next, check the integrity of the high molecular weight DNA once more by running out a small sample of the DNA using PFGE. The total amount of genomic DNA isolated from two grams of forced soil ranges from 10 to 180 micrograms as shown on the gel run before and after cesium chloride ultracentrifugation. The size range of the isolated DNA usually ranges between 40 to 60 kilobases shown here is a chromatogram of genomic DNA samples before and after cesium chloride ultracentrifugation.This step improves the purity of DNA increasing the two 60 over two 80 ratio from 1.3 to 1.6 before to 1.8 to 1.9 afterward. Moreover, the peak absorbance at 230 nanometers, which implies humic acid contamination was successfully reduced after centrifugation. We've just shown you how to isolate high molecular genomic DNA from soil microbial communities.When doing this procedure, it is important to remember to make the extraction and denaturing buffer precisely following the instruction. And be careful not to reach your DNA palate after isopropyl alcohol precipitation. When you recover the DNA band after chloride gradient centrification.Do not forget to rinse the syringe with the TE to maximize the DNA recovery. And finally, to check the quantity and quality of your isolated DNA at all suggested steps. So that's it.Thanks for watching and good luck with your experiment.

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Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

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