The overall goal of this procedure is to extract high molecular weight DNA from hyper saline microbial mats. This is accomplished by first homogenizing and extracting microbial cells from the microbial mat. The microbial cells are then lyed using freeze thaw and chemical methodology.
Next is the removal of cell debris and RNA using organic extractions and RNAs, followed by DNA extraction. The final step of the procedure is DNA precipitation and purification. Ultimately, results can be obtained that show DNA of high molecular weight quality and concentration.
The main advantage of this technique over existing methods, such as direct and harsh DNA extraction methods, is that it uses high molecular weight DNA from a complex environmental sample for functional metagenomic studies. The microbial met used in this study was obtained from a hyper saline pond located in Eleuthera, Bahamas to extract microbial cells from the background mat matrix first mix approximately 30 grams of mat thoroughly without grinding, with a sterile grinding pestle. The mat is thoroughly homogenized when no lumps can be seen in the mixture.
Next place the homogenized matte material into a blender and add about 100 milliliters of sodium chloride. Blend at medium speed for one minute, followed by cooling for one minute at minus 20 degrees Celsius. Repeat this, blending and cooling two more times when blending is complete, transfer the slurry into a 250 milliliter centrifuge bottle and fill the remaining empty volume with one molar sodium chloride.
Shake at room temperature for 30 minutes after 30 minutes, centrifuge the slurry at low speed for 15 minutes at four degrees celsius. Then gently transfer the supernatant into a flask with minimum disturbance to the sediment. Add fresh sodium chloride to the pelleted sediment.
Blend again and shake for 30 minutes. Then centrifuge as before, and transfer the supernatant into a new flask. Repeat the adding of fresh sodium chloride, blending centrifugation, and the removal of supernatant three additional times.
Combine the supernatants from the five cell extractions and centrifusion aliquots of 200 milliliters at high speed for 15 minutes at four degrees Celsius. Discard the supernatant and resuspend each cell pellet in 10 milliliters of sodium hexametaphosphate. Combine the cell suspensions from all four tubes and add sodium hexametaphosphate to make up the volume to 200 milliliters.
Wash cells by shaking at room temperature for 30 minutes. After shaking centrifuge cells at high speed for 15 minutes at four degrees Celsius, discard the supernatant and resuspend the cell pellet in 200 milliliters of te wash cells, again by shaking at room temperature for 10 minutes. Finally, centrifuge again at high speed for 15 minutes at four degrees Celsius.
After discarding the snat Resus, suspend the cell pellet in 15 milliliters of te prepare 200 microliter aliquots of microbial cells. Microbial cells can be stored at minus 80 degrees Celsius until needed for DNA extractions. To begin the procedure for extracting and purifying DNA from the microbial cells, add sodium chloride SDS and beamr capto ethanol to each of the 200 microliter microbial cell.
Aliquots eight tubes in total. Mix gently by inverting four to six times, submerge the mixture in liquid nitrogen for two minutes, followed by thawing at 65 degrees Celsius for five minutes, repeat the submersion in liquid nitrogen and thawing at 65 degrees Celsius. Two more times.
Extending the final thawing to 10 minutes. After the third round of freeze thaw, add 200 microliters of potassium acetate to each tube and place on ice for 10 minutes. Then centrifuge at 10, 000 GS for 10 minutes at four degrees Celsius.
Next, use a wide bore pipette tip to transfer the supernatant to a new tube. Add three microliters of RNAs A and incubate at 37 degrees Celsius for one hour. After the one hour incubation, add an equal volume of chloroform.
Shake briefly by inversion and centrifuge at 15, 000 Gs for 10 minutes. At room temperature, transfer the top layer of supernatant to a new tube. Repeat the chloroform extraction procedure.
Again, transfer the top layer of supernatant to a new tube. Add an equal volume of chilled isopropanol to the supernatant and incubate on ice for 30 minutes. To precipitate the DNA centrifuge at maximum speed, to pellet the DNA, discard the supernatant and wash each DNA pellet with one milliliter of chilled.70%ethanol.
Repeat the 70%ethanol wash centrifuge at max speed for 10 minutes at four degrees Celsius. Discard the supernatant. Repeat 70%ethanol wash and air dry DNA pellet for 10 minutes.
Resuspend each DNA pellet in approximately 25 microliters of te. Warm at 65 degrees Celsius for five minutes, and then combine into one tube and make up the volume to 500 microliters with te. Add polyethylene glycol mixed gently by inversion and incubate on ice for 10 minutes.
Centrifuge to pellet the DNA. Then remove and discard the supernatant after washing the DNA with chilled. 70%ethanol air dry for 10 minutes resuspend and 30 to 50 microliters of TE or molecular grade water and warm at 65 degrees Celsius for five minutes.
Store the DNA at minus 80 degrees Celsius. This table shows the results of measurement of concentration and quality of DNA extracted from the microbial hyper saline mat. The protocol yielded approximately two micrograms of DNA per gram of mat sample with an A two 60 to two 80 ratio of 1.6 and an A two 60 to two 30 ratio of 0.7.
Although the A two 60 to two 30 ratio appeared to be low, no inhibition of downstream molecular based applications such as PCR amplification of the 16 s ribosomal RNA genes was observed in a separate study when DNA size was determined using pulse field gel electrophoresis, the results indicate high molecular weight or H-M-W-D-N-A of approximately 35 to 50 kilobases. Here, lane M is an HMW marker and lanes one and two are replicates of metagenomic DNA extracted from the eleuthra hyper saline mat. The DNA smear above 35 kilobases may be excised and purified for large insert vector cloning or other appropriate molecular applications.
Following this procedure, other methods such as P-C-R-D-G-G-E cloning or DNA sequencing can be performed in order to answer additional questions like, what is the functional diversity of the microbial community under investigation.
