Koshland Web Site/Methods
E. Coli mini plasmid prep.
  1. Transfer 1-1.5 mls of the culture into an eppendorf tube.
  2. Spin 1 min. at 15K room temp. (remove with vacuum pump siphon)
  3. Resuspend in 300 µls of STET containing 1mg/ml lysozyme.
  4. Vortex, then Boil for 1 min. in the 100C temp block.
  5. Spin 10 min. at 15 K room temp.
  6. Remove and discard pellet using a tooth pick.
  7. Add 300 µls of cold Isopropanol, mix and leave at -20oC for 30 min. to 1 hour.
  8. Spin 15 min. at 15K 4oC. (remove supernatant with vacuum pump siphon)
  9. Wash with 70% EtOH. (remove supernatant with vacuum pump siphon)
  10. Dry pellets for 10 min. r/t. and resuspend in 50 µls of 1xTE containing 50µg/ml RNase.
  11. Incubate for 10 min. at 37oC.
  12. Analyse on agarose gel.

1x STET 100mls
8% Sucrose 8 gms Sucrose
50 mM Tris pH8.0 5 mls 1M Tris pH 8.0
50 mM EDTApH8.0 10 mls 0.5 M EDTA pH8.0
5% Triton X-100 5 mls Triton X-100
H2O to 100mls
for 18 samples prepare 6 mls of STET sol'n w/ 6 mgs of lysozyme

1XTE w/ 50µg/ml RNase : add 5µls of a 10 mg/ml RNase stock to 1ml 1XTE
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