Preparation of small quantities of yeast DNA
Yeast DNA can be prepared by digesting the cell wall and lysing the resulting protoplasts with SDS, and several milligrams of yeast DNA can be reproducibly prepared in this way. the yeast DNA obtained can be cleaved by restriction endonucleases and can be used as a template for PCR. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Preparation of small quantities of yeast DNA
Principle
Yeast DNA can be prepared by digesting the cell wall and then lysing the resulting protoplasts with SDS. several milligrams of yeast DNA can be reproducibly prepared in this way. the resulting yeast DNA can be cleaved by restriction endonucleases and can be used as a template for PCR.
Materials and Instruments
Enzymes 100T Yeast Cells Move I. Materials For more product details, please visit Aladdin Scientific website.
Isopropyl Alcohol Potassium Acetate SDS Sodium Acetate Sorbitol Buffer TE Yeast Resuspension Buffer
YFD Medium Sorvall SS-34 Turning Head or Equivalent Water Bath
1. Buffers and solutions
Isopropyl alcohol
Potassium acetate (5 mol/L )
SDS (10%, m/V)
Sodium acetate (3 mol/L, pH 7.0)
Sorbitol buffer
TE ( pH 7.4)
TE containing 20 μg/ml RNase (pH 8.0)
Yeast resuspension buffer
2. Enzyme and its buffer
Enzymes 100T
3. Culture medium
YFD medium
4. Centrifuges and their heads
Sorvall SS-34 head or equivalent
5. Specialized equipment
Water bath preset to 65°C
6. Carriers and yeast cell lines
Yeast cells
II. METHODS
Cell culture and its DNA extraction
1. 10 ml of YPD medium was used to culture yeast. The yeast culture was incubated overnight at 30°C with moderate shaking.
2. 5 ml of the cultured cells are placed in a centrifuge tube and centrifuged at 2000 g (Sorvall SS-34 head, 4100 r/min) for 5 min to collect the cells. The remaining culture was stored at 4℃.
3. Resuspend the cells with 0.5 ml sorbitol buffer. Transfer the cell suspension to a microcentrifuge tube.
4. Add 20 μl of ELISA 100T (2.5 mg/ml concentration in sorbitol buffer). Incubate the cell suspension for 1 h at 37 °C.
5. Collect the cells by centrifugation in a microcentrifuge for 1 min and discard the supernatant.
6. Resuspend the cells with 0.5 ml yeast resuspension buffer.
7. Add 50 μl of 10% SDS, cap the tube, turn the tube over rapidly several times, mix well and incubate the tube at 65°C for 30 min.
8. Add 0.2 ml of 5 mol/L potassium acetate solution and incubate on ice for 1 hour.
Isolation of DNA
9. Centrifuge the cells in a microcentrifuge at 4℃ for 5 min at maximum speed to deposit the cellular debris.
10. Aspirate the centrifugation supernatant with a coarse pipette tip at room temperature and transfer to a new microcentrifuge tube.
11. Add an equal volume of isopropanol at room temperature to precipitate nucleic acids. Mix the contents of the tube well and allow to stand at room temperature for 5 min.
12. Recover the nucleic acid precipitate by centrifugation at maximum speed for 10 s in a microcentrifuge. Aspirate the centrifugation supernatant and dry the precipitate in air for 10 min.
13. Dissolve the precipitate with 300 μl of TE (pH 8.0) containing 20 μg/ml tryptic RNase. The digested mixture was incubated at 37°C for 30 min.
14. Add 30 μl of 3 mol/L sodium acetate (pH 7.0). After mixing, add 0.2 ml of isopropanol. Mix again. Centrifuge in a microcentrifuge at maximum speed for 20 s to precipitate the DNA.
15. Aspirate the supernatant, dry the precipitate in air for 10 min, and dissolve the DNA with 150 μl TE (pH 7.0).