To begin, decant the 27.5 milliliters of the wastewater in 50 milliliter centrifuge tubes. Add 13.5 grams of polyethylene glycol 8000 and three grams of sodium chloride to the tubes and mix well until completely dissolved. Incubate the sample overnight at four degrees Celsius.
The next day, centrifuge the sample at 15, 500g for 30 minutes at four degrees Celsius. Discard the supernatant and dissolve the pellet in 800 microliters of the lysis solution from the soil DNA extraction kit. Then add the solution to the mixed zirconium bead tube.
Secure the bead tube horizontally on a vortex and vortex at maximum speed for 20 to 30 minutes. Spin down the tubes at 8, 000g for 15 seconds. After removing the froth, transfer the supernatant to a 1.5 milliliter micro centrifuge tube.
Centrifuge the supernatant at 15, 000g for one minute. Transfer the supernatant to a clean two milliliters micro centrifuge tube and add 200 microliters of precipitant solution. Vortex at maximum speed for five seconds before incubating at four degrees Celsius for five minutes.
Again, centrifuge the mixture and transfer the clear supernatant to a clean two milliliter micro centrifuge tube. Add 600 microliters of binding buffer per 700 microliters of supernatant. After vortexing, load 700 microliters of the lysate onto a silica spin column and incubate for two minutes.
Centrifuge the tube at 15, 000g for one minute and discard the flow-through. Now carefully place the spin column into a clean two milliliter collection tube. Add 500 microliters of wash buffer to the spin column and centrifuge at 15, 000g for one minute.
Discard the flow-through and return the spin column to the same two milliliter collection tube. Add 500 microliters of ethanol wash buffer to the spin column and centrifuge at 15, 000g for one minute. Discard the flow-through and place the spin column into a new two milliliter collection tube.
Centrifuge at 16, 000g for two minutes to remove residual ethanol. Place the spin column into a 1.5 milliliter tube. Add 100 microliters of preheated nuclease-free water to the center of the white filter membrane and incubate for five minutes.
Then centrifuge the tube at 15, 000g for one minute. To the eluted DNA, add 10 microliters of three molar sodium chloride and 250 microliters of chilled absolute ethanol and invert to mix well. Spin down the contents at 8, 000g for 15 seconds.
Incubate the DNA ethanol mixture at minus 20 degrees Celsius for one hour. Then centrifuge the mixture at 19, 000g for 30 minutes at four degrees Celsius and decant the tube to discard the supernatant. Add 500 microliters of chilled 70%ethanol to the pellet and centrifuge at 19, 000g for 15 minutes at four degrees Celsius.
Decant the supernatant and gently tap the tube on tissue paper to remove residual ethanol. Then place the tubes open on a heating block for five to 10 minutes at 37 degrees Celsius to dry the DNA pellet. Resuspend the pellet in 30 to 50 microliters of nuclease-free water and incubate at 37 degrees Celsius for five to 10 minutes.
Spin down the DNA at 8, 000g for 15 seconds. On a NanoDrop spectrophotometer under the nucleic acid settings, select the double stranded DNA application. Clean the pedestal with lint-free tissue paper and water.
Use nuclease-free water to blank the instrument, then wipe the pedestal and load two microliters of the DNA sample on it. Measure the concentration and absorbance ratios to determine the purity. After measurement, store the sample at minus 20 degrees Celsius.
