which contains amber mutated ampicillin and tetracycline drug resistance elements

(Seed, 1983). The p3 plasmid id derived from RP1, is 57kb in length, and is a stably

maintained, single copy episome. The ampicillin resistance of this plasmid reverts at a

high rate, so that ampr plasmids usually cannot be used in p3-containing strains.

Swlwction for tet resistance alone is almost as good as selection for amp+tet resistance.

However spontaneous appearance of chromosomal suppressor tRNA mutations presents

an unavoindable background (frequence about 10 -9) in this system. Colonies arising from

spontaneous suppressor mutations are usually bigger than colonies arising from plasmid

transformation. We typically select for suppressor plasmids in LB medium containg

amp at 12.5 ug/ml and tet at 7.5 ug/ml/ For large plasmid preps we use M9 casamino acids

medium containing glycerol (0.8%) as a carbon source, and grow the bacteria to saturation.