Protocols

Plasmid Extraction from Yeast (Glassbeads-Column)

  1. Grow 10 mL culture to saturation. Harvest by centrifugation. Decant supernatant and resuspend cells in residual liquid. Transfer cells to screw-cap microcentrifuge tube.
  2. Spin 2 min (13 000 rpm) in microfuge. Discard supernatant.
  3. Add 100 µL of Lysis buffer, 100 uL of phenol:chloroforme (50:50) and acid-washed glass beads. Vortex 5-10 min.
  4. Add 100 µL of TE (pH 8). Microfuge 8 min (13 000 rpm). Transfer aqueous layer to fresh tube (~200 µL).
  5. Add an equal volume of chloroform. Mix by inversion, microfuge 2 min (13 000 rpm). Transfer aqueous layer to fresh tube.
  6. Add an equal volume of buffer N3, mix by inversion.
  7. Transfer the lysate to QIAprep Spin Column. Centrifuge 30-60s (13 000 rpm). Discard flow-through.
  8. Wash QIAprep Spin Column by adding 750 µL of Buffer PE and centrifuging 30-60s (13 000 rpm).
  9. Discard flow-through and centrifuge for an additional 1 min to remove residual wash buffer (13 000 rpm).
  10. Place QIAprep Spin Column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 uL of water to the center of each column, and centrifuge for 1 min.
Protocole from :
Preparing Chromosomal DNA (alternative method); Hoffman & Winston; Gene 57, 267-272 (1987)
et Isolation of plasmid DNA from yeast using the QIAprep Spin Miniprep Kit; www.qiagene.com/literature/handbooks/default.asp