Growth of P3 plasmid E.Coli

Dominik Haudenschild, November 8, 1994, at CBRC from Invitrogen Protocol

History

P3 plasmid is a low copy 60kb episome which carries the drug resistance markers Kanamycin, Tetracycline, and Ampicillin. The Tetracycline and amp genes on the P3 plasmid contain amber mutations which render them inactive during normal growth. Kanamycin gene is fully active and can be used to select for cells carrying the P3. Upon introduction of plasmid vector carrying the suppresser-F gene (Sup-F) such as pcDNA-I and pCDM8, the amber mutations in the he Tetracycline and amp genes on the P3 plasmid are suppressed, and the E.coli becomes resistant to those antibiotics.

The drug resistance markers with amber mutations spontaneously revert. The reversion rate on Amp is about 5% and on Tetracycline is about 1%. It is important to reduce antibiotic levels to allow cells containing supF plasmids sufficient time to suppress the amber mutations on P3. Use Tetracycline at 7.5 to 10µg per ml, and Ampicillin at 25 to 40µg per ml.

Select P3 containing colonies as follows:

1) remove bacteria from glycerol stock and disperse into 250µl of LB

2) Streak these bacteria from liquid media onto agar plates containing 40µg/ml Kanamycin to select for P3 plasmid.

3) Following overnight incubation at 37°C spot several Kanamycin resistant colonies onto plates that contain Tetracycline (7.5µg/ml) and Ampicillin (30µg/ml). Also spot the same colonies onto a fresh Kanamycin plate.

4) Following overnight incubation at 37°C, pick colonies from the Kanamycin plate that exhibit sensitivity to Tetracycline and Ampicillin. Use one of these colonies to prepare competent cells.