Yeast Chromosomal DNA Prep
- use 25 ml cells per timepoint / sample
- add cells to 25 ml 100 % Ethanol (at -20 °C), add 1 ml 0.5 M EDTA
- store at -20 °C
- thaw cells, wash once with H2O, resuspend in 0.5 ml Shperoplast buffer
- incubate for 30 min at 37 °C
- add 0.5 ml Proteinase K buffer
- incubate for 30 min at 65 °C
- add 0.2 ml 5 M KAc, incubate on ice for 10 min
- spin at 10,000 rpm for 15 min
- take supernatant and Ethanol precipitate the DNA
- resuspend DNA in 500 µl TE
- RNaseI digest for 30 min at 37 °C
- extract with Phenol/Chloroform
- Ethanol precipitate DNA
- dissolve DNA in TE and measure concentration
- use 2-5 µg for Southern
blot
Buffers:
Shperoplast buffer:
1.2 M sorbitol; 0.1 M EDTA; 1 % b-Mercaptoethanol; 0.1 % Zymolyase.
[ for 10 ml: 6 ml 2 M sorbitol; 2 ml 0.5 M EDTA; 100 µl
b-Mercaptoethanol; 10 mg Zymolyase]
42 °C Proteinase K buffer:
50 mM EDTA; 0.3 % SDS; 0.01 % proteinase K.
[for 10 ml: 1 ml 0.5 M EDTA; 300 µl 10 % SDS; 1 mg
proteinase K]
