Isolation of mt DNA from Yeast

 

Reagents

10mg/ml Bisbenzimide in TE

 

Z- buffer:

1.2M sorbitol

50mM NaPi, pH 7.5

25mM EDTA

1% 2-ME

 

B-Buffer

0.2M Tris-Cl, pH 9.4

80mM EDTA

1% SDS

 

D-buffer                     

25mM EDTA

50mM Pi, pH 7.5

 

TE

10mM Tris-Cl, pH 7.5

1mM EDTA

 

Procedure

 

  1. Grow 3 x 1.5 liters of yeast in YPGal with 0.5% Glucose ON.
  2. Spin 5000 rpm for 10 min.  Wash 1x with cold water  (about 40gms yeast).
  3. Suspend in 100 ml of Z-buffer with 100 mg of Zymolase 20T.
  4. Incubate at 37oC for 1-2 hours.  The yeast should be sticky to the glass wall.  Check under microscope for lysis in 1% SDS.
  5. Add 100 ml of cold D-buffer, with stirring.
  6. Homogenize 5 strokes.
  7. Spin at 5000 x g for 10 min. at 4oC.
  8. Spin supernatant at 18,000 rpm for 20 min.,
  9. Collect pellet and resuspend in 50 ml B-buffer.
  10. Incubate at rt  for 10 min.
  11. Add 25ml of 5M KAc.  See Maniatis for instructions on how to make this.
  12. Cool on ice for 45 min.
  13. Spin at 18,000 rpm in SS-34 for 45 min at 4oC.
  14. Add 1 vol of 2-propanol to supernatant.
  15. Let sit ON at 4oC.
  16. Spin at 15k x g for 15 min.
  17. Take pellet and dissolve in 10 ml of TE
  18. Add 0.1 ml of 10mg/ml Bisbenzimide
  19. Add 11.8 g CsCl
  20. Adjust RI to 1.398
  21. Add to heat sealable tubes for VTi65.1. Spin for 18 hours at 15oC at 49,000 rpm.  Brake is set to off or slow.
  22. Take the top band as seen under long wavelength UV light (360nm).
  23. Repeat spin a by adding more CsCl/Bisbenzimide solution and repeating steps 21-22 (optional).
  24. Extract (3 x) with 2-propanol saturated with CsCl solution.
  25.  Add 4 volumes of 0.3M NaAc, pH 5.5.  Mix and add 3 volumes of ETOH.  Freeze at –80oC for 5 min. or 20oC ON.
  26. Spin 8K rpm for 20 min in a glass Corex tube. 
  27. Dissolve in TE and add NaAc to 0.3M, and 2 vol ETOH, and ppt again.
  28. Collect as above.
  29. Cut with EcoRI and separate by 1% agarose gel to check purity.