Growth of Saccharomyces cerevisiae and its DNA preparation
DNA prepared by this method is suitable for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that do not require intact high molecular mass DNA. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Growth of Saccharomyces cerevisiae and preparation of its DNA
Principle
DNA prepared by this method is suitable for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that do not require intact, high molecular mass DNA.
Materials and Instruments
YAC Cloned yeast colonies Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate Ethanol Phenol Chloroform TE Triton SDS solution
YPD medium Sorvall SS-34 turn head or equivalent Glass beads
1. Buffers and solutions
Ammonium acetate (10 mol/L )
Ethanol
Phenol: chloroform (1:1, V/V)
TE ( pH 8.0)
TE with 20 μg/ml RNase (pH 8.0)
Triton/SDS solution
2. culture medium
YPD medium
3. Centrifuges and rotors
Sorvall SS-34 Turntable or equivalent
4. Specialized equipment
Glass beads
5. Carriers and yeast strains
Yeast colonies carrying the target YAC clone
II. METHODS
Cell culture
1. Inoculate a yeast colony containing the target YAC clone into 10 ml of YPD medium and shake at 30℃ overnight.
2. Collect the cells by centrifugation at 2000 g (Sorvall SS-34 head, 4100 r/min) for 5 min.
3. Discard the medium, add 1 ml of sterile water and shake gently to suspend the cells in the solution.
4. Collect the cells as in step 2.
5. Remove the supernatant, resuspend the cells in 0.5 ml of sterile water and transfer to a 1.5 ml sterile centrifuge tube.
6. Centrifuge in a microcentrifuge at maximum speed for 5 s at room temperature, remove the supernatant and collect the cells.
DNA extraction
7. Add 0.2 ml of Triton/SDS solution to the cells and tap the side of the centrifuge tube to resuspend the cell sediment.
8. Add 0.2 ml of phenol: chloroform and 0.3 g of glass beads and shake at room temperature for 2 min. Shake at room temperature for 2 min. add 0.2 ml TE (pH 8.0) and shake briefly to mix the system.
9. Separate the organic phase from the aqueous phase by centrifugation in a microcentrifuge at maximum speed for 5 min at room temperature. Transfer the upper aqueous phase into a new centrifuge tube. Handle with care to avoid aspiration of material at the interface of the two phases.
Isolation of DNA
10. Add 1 ml of ethanol to the aqueous phase, cap the tube, gently turn the tube over several times and mix well.
11. Centrifuge the sample in a microcentrifuge at maximum speed at 4℃ for 2~5 min to precipitate the DNA. Remove the supernatant with a Pasteur pipette. Centrifuge again briefly (2 s ) to remove the last remaining ethanol from the bottom of the tube.
12. Dissolve the nucleic acid precipitate with 0.4 ml of TE (pH 8.0) containing RNase and incubate at 37°C for 5 min.
13. Extract the RNAase-digested solution by adding an equal volume of phenol/chloroform. It is best to mix the solution by turning the tube over, not by vortexing. Centrifuge in a microcentrifuge at maximum speed for 5 min at room temperature to separate the aqueous and organic phases. Transfer the aqueous phase to another new centrifuge tube.
14. Add 80 μl of 10 mol/L ammonium acetate and 1 ml of ethanol to the aqueous phase. Gently turn the tube over to mix. Allow to stand at room temperature for 5 min.
15. Collect the DNA precipitate by centrifugation in a microcentrifuge for 5 min. Discard the supernatant and rinse the nucleic acid precipitate with 0.5 ml of 70% ethanol. Centrifuge at maximum speed for 2 min and remove the ethanol rinse with a Pasteur pipette. Centrifuge again briefly (2 s) and aspirate the last ethanol residue from the bottom of the tube. The DNA precipitate was dried in air for 5 min and dissolved in 50 μl TE (pH 8.0).