Under non-selective growth conditions, most E. coli/yeast shuttle vectors are lost at a frequency of about 1%, and one plasmid that is not easily isolated and removed using this scheme is the endogenous 2 μm plasmid, with spontaneous loss of 2 μm DNA at a frequency of about 10-4 per generation.

Operation method

basic program

Materials and Instruments

Yeast
YPD
Inoculation needle Culture flask Shaker

Move

1. Pick a few plasmid-containing colonies of yeast host bacteria, inoculate them in 10 ml of non-selective medium, and incubate them at 30°C overnight.2. Incubate on non-selective plates at 30°C for 2 days to obtain single colonies, in most cases about 200-300 single colonies can be obtained (about 100 colonies per plate).
3. Identify colonies that have lost the plasmid selection marker by photocopying onto a selective plate and incubating overnight at 30°C. Those colonies that can grow on the master plate but not on the selective plate have lost the plasmid.

Caveat

YPD medium can be used if there are no other options, or if other unstable genetic markers or plasmids are to be retained, defined component medium with added nutrients corresponding to the selection markers that may be lost can be used.

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