Yeast DNA Preparation for Recovery of BAC DNA into Bacteria

Adapted from Boeke et al. (1985).

Protocol:

  1. Inoculate a single yeast colony into 5-6 ml YPD (or appropriate media) and grow overnight, shaking, at 30°C.

  2. Reserve ~1 ml to make a freezer stock of your saturated yeast culture. With the remainder (or just 3 ml usually), centrifuge culture at 3600 rpm for 5 min at 4°C to harvest cells.

  3. Resuspend cells in 1 mL Solution I.

  4. Transfer cells to an eppendorf tube.

  5. Centrifuge cells at 3600 rpm for 3 min at room temperature.

  6. Resuspend cells in 400 μl Solution II.

  7. Add 100 μl Zymolyase Solution.

  8. Incubate at 37°C for 30 minutes. (Note: 30 minutes is an approximate time; monitor spheroplast formation under a microscope.)

  9. Centrifuge at top speed for 20 sec.

  10. Resuspend cells in 400 μl TE by pipetting.

  11. Add 90 μl Solution III and mix well.

  12. Incubate at 65°C for 30 min.

  13. Add 80 μl 5M Potassium Acetate and mix well.

  14. Incubate on ice for one hour.

  15. Centrifuge at top speed for 15 min. at room temperature.

  16. Transfer supernatant to a new tube.

  17. Add 1 ml ethanol and invert by mixing.

  18. Centrifuge at top speed for 30 sec.

  19. Wash pellet with cold 70% ethanol.

  20. Air dry pellet 5-10 min.

  21. Resuspend in 500 μl TE. Tap tube to resuspend DNA, do not vortex as this will shear the DNA. (Note: can store at 4°C overnight)

  22. Centrifuge at top speed for 15 min.

  23. Transfer supernatant to a new tube.

  24. Add 15 μl 2 mg/ml RNase A.

  25. Incubate at 37°C for 30 min.

  26. Add 500 μl 25:24:1 phenol/chloroform/isoamyl alcohol.

  27. Rotate to mix for 30 min at room temperature.

  28. Centrifuge at top speed for 10 min.

  29. Draw off 400 μl of aqueous phase using wide bore tips, and transfer to a new eppendorf tube.

  30. Add 500 μl isopropanol and mix gently by inverting.

  31. Centrifuge at top speed for 30 sec.

  32. Wash pellet with 70% ethanol.

  33. Air dry pellet 5-10 min.

  34. Resuspend DNA in 30-40 μl water – has to be salt free for downstream electroporation.

  35. Transform 1-2 μl into electrocompetent cells. (Note: ElectroMAXTM DH10BTMT1 'Phage-Resistant Competent Cells from Invitrogen, Catalog number 12033-015, work very well for transformation of BAC DNA.)


Solutions:

Solution I

  • 1 M Sorbitol

  • 0.1 M EDTA

  • pH 7.5

Solution II

  • 0.9 M Sorbitol

  • 0.1 M EDTA

  • 14 mM β-mercaptoethanol

Zymolyase

  • 2 mg/ml zymolyase 100T

  • 1 M Sorbitol

Solution III

  • 1.5 ml 0.5 M EDTA

  • 0.6 ml 2 M Tris

  • 0.6 ml 10% (w/v) SDS

  • pH 8.0