The yeast is first lysed using a solution containing leaving salts to ensure complete denaturation of the macromolecules. Afterwards, ethanol is added and the DNA binds to the silica gel mold of the centrifuge column as the lysate is centrifuged through the microcentrifuge tube. After washing to remove contaminants, the DNA is eluted in Tris-EDTA solution. (The recovered DNA can be used for digestion, PCR, Southern hybridization, etc.).
Operation method
Yeast Genome DNA Extraction
Materials and Instruments
Yeast Genomic DNA Extraction Kit (Kit contains: Buffer GA, Buffer GB, Buffer GD, Move Add anhydrous ethanol to buffer GD and rinse solution PW before use, and refer to the instructions of the kit for the volume to be added. 1, take 1.5 ml of fresh yeast liquid, centrifuge at 12 000 rpm for 1 min, and try to absorb the supernatant as much as possible. 2、Break down the yeast cell wall: add 600 μl sorbitol buffer to the yeast, add about 50 U Lyticase, mix thoroughly. 30 minutes at 30 ℃, centrifuge at 4 000 rpm for 10 minutes, discard the supernatant, and collect the precipitate. 3. Add 200 μl of GA buffer to the precipitate, resuspend the precipitate and mix well. 4. Add 20 μl of Proteinase K solution and mix well. 5, add 220 μl of buffer GB, mix thoroughly upside down, 70 ℃ for 10 min, the solution should become clear, briefly centrifuged to remove the water droplets on the inner wall of the cap. 6, add 220 μl of anhydrous ethanol, mix thoroughly inverted, at this time may appear flocculent precipitation, briefly centrifuged to remove the water droplets on the inner wall of the cap. 7: Add the solution from the previous step and the flocculent precipitate to an adsorbent column CB3 (adsorbent column in a collection tube), centrifuge at 12,000 rpm for 30 sec, pour off the waste liquid, and put the adsorbent column CB3 into a collection tube. 8. Add 500 μl of buffer GD to column CB3 (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 sec, pour off the waste solution, and place the column CB3 into a collection tube. 9. Add 600 μl of Rinse Solution PW (check that anhydrous ethanol has been added before use) to the column CB3, centrifuge at 12 000 rpm for 30 sec, pour off the waste solution, and place the column CB3 into a collection tube. Repeat the procedure once. 10. Place the column CB3 back into the collection tube, centrifuge at 12 000 rpm for 2 min, and pour off the waste liquid. Leave the column CB3 at room temperature for 5 minutes to thoroughly dry any rinse solution remaining in the adsorbent material. 11. Transfer the adsorbent column CB3 into a clean centrifuge tube, add 50~200 μl of elution buffer TE dropwise to the middle of the adsorbent membrane, leave it at room temperature for 5 min, centrifuge it at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube. 12, Nanodrop 2 000 to determine the genomic concentration, -20 ℃ storage.
Buffer PW, Buffer TE, Proteinase K, adsorption column CB3, 2 ml collection tube), Lyticase, Sorbitol Buffer.
Instruments: Centrifuge
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