This is a cached page for the URL (http://www.fhcrc.org/labs/breeden/Methods/chromosomal-DNA.html). To see the most recent version of this page, please click here. Protocol Online is not affiliated with the authors of this page nor responsible for its content. About Cache

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]

---