S. pombe Genomic DNA Miniprep (glass beads method)

Note: We don’t have a FastPrep instrument, but the Iyer or Marcotte labs might have one.

Contents

1. Materials
2. Protocol

1. Materials

STEN buffer : 0.1 M Tris pH 8.0 0.1 M NaCl 1 mM EDTA, pH 8.0 1 % SDS

• For 100 ml of STEN Buffer:
  • 10 ml of 1 M Tris, pH 8.0
  • 2 ml of 5 M NaCl
  • 20 ml of 0.5 M EDTA pH 8,0
  • 200 µl of SDS 20%
• PCI [Phenol/Chloroform/Isoamyl Alcohol (25:24:1v/v)]
• TE 1X
• 70% and 100% Ethanol
• Glass Beads, acid washed 425-600 µm (Sigma #G8772)

2. Protocol

1. Inoculate one S. pombe colony in 5 ml of YES at 30°C until stationary phase (OD600~2).
2. Spin the cells for 5 min at 5000 rpm and resuspend in 250 µl of STEN buffer.
3. Transfer the supernatant in a screw cap 1.5 ml tube filled up to 250 µl with glass beads. Add 250 µl of PCI. Break for 30 sec at speed 5 in the FastPrep instrument.
4. Spin for 3 min at 15,000 rpm and take 225 µl of the aqueous phase to a 1.5 ml Eppendorf tube. Add TE up to 500 µl.
5. Repeat the PCI extraction and take 400 µl of the aqueous phase to a new tube.
6. Add 2 volumes of ethanol, spin 5 min at 15,000 rpm.
7. Wash the DNA pellet with 500 ml of ice-cold 70% ethanol.
8. Let the pellet dry.
9. Resuspend the pellet in 100 µl of TE at 20 µg/ml of RNase A.