Fast small scale isolation of total yeast DNA (Hoffman and Winston, 1987)

Reference:

Hoffman C.S. and Winston F., 1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267-272

Protocol

  1. Prepare an o/n culture (10 ml YPD+ or SD) at 30°C
  2. Harvest cells by centrifugation (5 min, 3500 rpm)
  3. Resuspend cells in 0,5 ml water
    Transfer into Eppendorftube
    Collect cells by centrifugation (5 sec at 13000 rpm)
  4. Resuspend cell pellet in 200 µl buffer 1
    Add 0,3 g glass beads (acid treated, 0,45-0,51 mm) (i.e., the same volume as the cell suspension)
  5. Add 200 µl phenol/chloroform/isoamylalcohol (25/24/1)
  6. Place tubes in mixer mill for 3 min
  7. Add 200 µl buffer TE
    Vortex
  8. Centrifuge for 5 min at 13.000 rpm
  9. Transfer water phase into clean Eppendorf tube
  10. Add 1 ml of Ethanol (p.a.)
  11. Spin down for 2 min at 13.000 rpm
  12. Resuspend pellet in 400 µl TE
  13. Add 30 µg RNaseA
  14. Incubate for 5 min at 37°C
  15. Add 10 µl NH4Oac 5 M
    Add 1 ml of Ethanol (p.a.)
  16. Centrifuge for 2 min at 13.000 rpm
  17. Wash DNA pellet with EtOH (70%)
  18. Dry DNA pellet
  19. Resuspend pellet in 50 µl TE

Materials

Buffer 1: For 50 ml
2 % Triton-X100 10 ml 10% Triton-X100
1% SDS 2,5 ml 20% SDS
100 mM NaCl 1 ml 5 M NaCl
10 mM Tris.HCl pH 8,0 0,5 ml 1 M Tris.HCl pH 8,0
1 mM EDTA 0,1 ml 0,5 M EDTA pH 8,0

 

Preparation of glass beads:
soak in concentrated nitric acid for 1 h
Wash extensively with water
Bake until dry

Remark

If plasmid DNA is to be prepared for transformation of E. coli, steps 12-16 of the protocol can be skipped. Resuspend the final DNA pellet in 5-10 µl of TE buffer, and use 1,5 µl to electroporate E. coli XL1-Blue cells. An average of 50-100 transformants can be expected.

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