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.
