Overview[1]
Phenol-chloroform-isoamyl alcohol mixture is also called phenol-chloroform-isoamyl alcohol mixture, which refers to a mixture composed of the above three reagents.
Apply[1]
The method for efficiently extracting DNA from soil microorganisms includes the following steps:
(1) Weigh 0.3-0.4g of sample and add it to Lysing Matrix E Tube. Add 978μl of SPB and 122μl of MT Buffer;
(2) Tightly close the lid of the Lysing Matrix E Tube, place the centrifuge tube on the vortex mixer, and shake, crush, and homogenize the contents of the tube. The time is set to 40-50 seconds. Centrifuge at 8000r/min for 15min.
(3) Use a large pipette tip to suck out the supernatant and transfer it into a 2ml microcentrifuge tube. Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), mix by inversion, and set at 15°C. 10000rpm, centrifuge for 10min;
(4) Transfer the supernatant to a clean centrifuge tube, add an equal volume of chloroform-isoamyl alcohol (24:1), mix by inverting, centrifuge at 15°C, 10000rpm for 10 minutes, and take the supernatant;
(5) Add 250μl of PPS, mix by inverting more than 10 times, and let stand at room temperature for 10 minutes;
(6) Centrifuge at 8000r/min for 5 minutes, and use a large pipe tip to transfer the supernatant to a 2ml centrifuge tube;
(7) Take 800μl of the shaken Binding Matrix and add it to the centrifuge tube in the previous step;
(8) Turn the centrifuge tube upside down for 2 minutes and then let it sit for 3 minutes, waiting for the silica matrix to precipitate;
(9) Remove 500μl supernatant to avoid aspirating the sediment;
(10) Mix the remaining supernatant and sediment again, transfer about 600μl of the mixed solution into the SPIN Filter, and centrifuge at 8000r/min for 1 minute;
(11) Pour out the liquid in the collection tube and repeat the previous step until the liquid in the 2ml centrifuge tube is exhausted;
(12) Add 500μl of the prepared SEWS-M solution, mix carefully with a small pipette tip, then centrifuge at 8000r/min for 1 minute, and discard the liquid in the collection tube;
(13) Repeat the previous step and replace the collection tube with a 1.5ml Catch Tube;
(14) Air-dry the SPIN Filter at room temperature for 5-10 minutes;
(15) Add 80μl of DES solution to the SPIN Filter, use a small pipette tip to mix it with the Binding Matrix, and place in a 65°C water bath for 15 minutes. Centrifuge at 8000r/min for 2 minutes to obtain about 50 μl of DNA solution. Then add 80 μl of DES solution and repeat this step to obtain about 100 μl of soil microbial DNA solution.
Main reference materials
[1] [Invented in China] CN201711361715.X A method for efficiently extracting soil microbial DNA
