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Yeast gDNA Isolation Midi/Maxi Kit(Solution)

Packing Specification：

Product Introduction：
This kit is used for the rapid extraction of genomic DNA from yeast. Under the action of Yeast Lysis Buffer formulated for the characteristics of yeast cells, yeast cells are lysed to release genomic DNA. Then Protein Precipitation Buffer selectively precipitates and removes proteins. Finally, pure genomic DNA is precipitated with isopropanol and redissolved in DNA Dissolution Buffer.

Application Scope：
Suitable for the rapid extraction of genomic DNA from yeast.

Product Features：
1.No need for toxic reagents such as phenol or chloroform.
2.Fast and simple; the entire process can be completed within 1 hour.
3.Stable results and high yield (more than twice the yield of spin column-based kits). The typical OD260/OD280 ratio is 1.7-1.9, and the DNA length can reach 50 kb-150 kb. It can be directly used for PCR, Southern blot, various restriction enzyme digestion reactions, and library construction.

Operation Steps：
1.Pipette 60 mL-70 mL of yeast culture into a 100 mL centrifuge tube. Centrifuge at 2,500 x g for 2 minutes. Discard the supernatant as much as possible, aspirating with a pipette if necessary.

2.Vortex vigorously to disperse and resuspend the yeast cell pellet.

3.Add 20 mL of Yeast Lysis Buffer, vortex to mix, or repeatedly pipette with a 1 mL pipette tip to mix thoroughly.
Thorough resuspension and dispersion of yeast cells are critical for the next lysis step and must be fully achieved.

4.Incubate the lysate in a 70℃ water bath for 15-30 minutes.
If the yield is low, appropriately increase the water bath temperature and extend the incubation time. Vortex to mix several times during incubation to assist lysis.

5.Place on ice for at least 5 minutes to return to room temperature.

6.Add 6.8 mL of Protein Precipitation Buffer to the room temperature lysate, then vortex continuously at high speed for 25 seconds. Small protein clumps may be visible after mixing. Incubate on ice for 5 minutes.

7.Centrifuge at 2,500 x g (centrifugal force can be increased if needed) for 5 minutes. A protein pellet should be visible at the bottom of the tube, and some protein precipitate may also float on the liquid surface.

8.Carefully and slowly aspirate the supernatant into a new 100 mL centrifuge tube, avoiding the precipitate.
Do not aspirate the protein pellet at the bottom or floating on the surface. If protein precipitate is accidentally transferred to the new tube, centrifuge again for 2 minutes and collect the supernatant.

9.Add an equal volume of room temperature isopropanol, invert 30 times to mix until flocculent DNA precipitate (or white turbid precipitate) appears.

10.Centrifuge at 2,500 x g for 5 minutes (centrifugal force can be increased if needed). A white DNA pellet will be visible at the bottom of the tube. Discard the supernatant.

11.Add 20 mL of 70% ethanol, invert several times to wash the DNA precipitate, then centrifuge at 2,500 x g for 2 minutes. Discard the supernatant (be careful not to discard the DNA pellet). Invert the tube and tap gently on absorbent paper to drain residual ethanol, or carefully aspirate residual ethanol around the pellet and tube wall with a pipette tip. Air-dry the pellet for a few minutes.
Avoid over-drying (DNA will be extremely difficult to dissolve) or leaving excessive residual ethanol (which may inhibit downstream reactions such as enzyme digestion).

12.Add 1-3 mL of DNA Dissolution Buffer to rehydrate and dissolve the DNA pellet. Tap the tube wall to mix. Incubate at 65℃ for 30-60 minutes (no more than 1 hour), or allow hydration at room temperature or 4℃ overnight, tapping the tube wall occasionally to assist hydration.

13.Add 0.5-1 mL of RNase A (10 mg/mL), invert to mix, and incubate at 37℃ for 30-60 minutes to remove residual RNA.
This step is mainly to remove residual RNA. If there is a large amount of residual RNA, the incubation time can be appropriately extended or the dosage of RNase A can be increased. If residual RNA does not affect the experiment, this step can be omitted. If residual RNase may interfere with the experiment, the RNA can also be removed by extraction with an equal volume of phenol/chloroform, followed by standard ethanol precipitation to recover DNA.

14.Store DNA at 2-8℃ for short-term use or -20℃ for long-term storage.