Dominik Haudenschild August 10, 1993 at CBRC

1) Grow an overnight culture in LB with appropriate antibiotic.

2) Pellet cells in 1.5 ml eppendorf tube @ max. speed for 2 min.

3) Resuspend cell pellet in 200 µl of STET buffer.

4) Add 4 µl of 50 mg/ml lysozyme, and incubate @ RT for 5 min..

5) Put sample in boiling water bath for 45 sec.

6) Centrifuge in eppendorf at top speed for 10 min.

7) Remove the chromosomal DNA pellet with a toothpick.

8) Add 5 µl of 10 mg/ml RNase A (that is DNase free!!), and incubate @ 68° C for 10 min.

9) Add 10 µl of 5% W/V CTAB, centrifuge for 5 min.

10) Discard Supernatant.

11) Resuspend pellet in 300 µl of 1.2 M NaCl. This requires vortexing.

12) Precipitate the nucleic acids with 750 µl of ethanol, and centrifuging for 5 min.

13) Wash the final pellet with 70% ethanol and resuspend in 20 µl of TE (10:1, pH7-8). The average yield for Bluescript plasmids is ~5 µg.

STET Buffer

8% Sucrose

50 mM tris, pH 8.0

50 mM EDTA

0.1% V/V TritonX-100

5% W/V CTAB (Sigma Chemical Co.)

0.5 M NaCl

Reference: G. Del Sal, G. Manfioletti and C. Schneider (1989). The CTAB-DNA Precipitation Method: A Common Mini-Scale Precipitation of Template DNA From Phagemids, Phages or Plasmids Suitable for Sequencing. Biotechniques 7:514-519.