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Yeast Chromosomal DNA Prep

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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 H2 O, 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]

 

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