To begin, take 100 milligrams of fresh tissue or 20 milligrams of air-dried tissue and place it in a two-milliliter centrifuge tube. Place two five-millimeter stainless steel beads into the centrifuge tube and transfer it to a high-throughput tissue grinder operating at 60 hertz for 60 seconds. For DNA extraction, add 800 microliters of pre-cooled nuclear isolation buffer to the sample and place it on a vortex mixer for five minutes.
Then, place the sample in a centrifuge and spin at 13, 780g for 10 minutes. After centrifugation, remove the supernatant carefully. Next, add 600 microliters of Buffer P1, followed by five microliters of RNase A to the sample.
After vortexing, incubate the samples in a water bath at 65 degrees Celsius for 1.5 hours, inverting the tubes two to three times during incubation. Then, add 200 microliters of Buffer P2.Mix thoroughly. And place the sample on ice for 15 minutes.
Afterward, centrifuge at 13, 780g for 10 minutes. Carefully aspirate the supernatant onto a filter column AF and centrifuge it at 13, 780g for two minutes. Transfer the lower filtrate to a new 1.5-milliliter centrifuge tube.
After calculating the volume of the filtrate, add 1.5 times the volume of Buffer P3 to the sample and shake immediately to mix the sample. Add 650 microliters of the prepared mixture to an absorption column and incubate for five minutes. Then, centrifuge at 13, 780g for 30 to 60 seconds.
Add the obtained filtrate into the absorption column again. Centrifuge and discard the waste liquid. Wash the column twice with 600 microliters of rinse solution WB.After centrifugation, discard the waste liquid, then place the absorption column into an empty collection tube.
After centrifuging the sample at 13, 780g for two minutes, leave it at room temperature to evaporate residual ethanol. Transfer the absorption column into a clean 1.5-milliliter centrifuge tube, and add 45 microliters of sterilized deionized water preheated to 65 degrees Celsius to the center of the adsorbent membrane. Five minutes later, centrifuge the tube at 13, 780g for two minutes at room temperature.
Then, use a spectrophotometer. Determine the DNA concentration in the filtered solution. The absorbance ratios of OD260 to OD280 ranged from 1.8 to 1.84, and the DNA quantity exceeded 100 nanogram per microliter, confirming good quality of the extracted DNA.
