FAQ: What guidelines are recommended for improving clone stability?

There are two primary causes of clone instability: 1) the plasmid construct contains repetitive DNA sequences or 2) cloned gene products are toxic to the bacterial cell. High copy cloning vectors with a pMB1 origin of replication that lacks the rop gene (e.g. pUC19 and many common vectors) will replicate at a lower copy number per cell as the temperature is decreased. Thus for greatest clone stability, culture cells at 30°C or below and also incubate transformation plates at 30°C or below. The lacIq gene present within NEB Stable cells overexpresses LacI repressor to minimize expression of toxic genes cloned into polylinkers capable of alpha-complementation (containing the lac promoter/operator).

Also for best clone stability, prepare plasmid DNA from fresh transformants (from plates not greater than three days old). Store unstable clones as plasmid DNA, rather than cell-based glycerol stocks.

Gibson Assembly cloning (#E2611 – Gibson Assembly Master Mix) is an especially efficient method for isolating difficult clones. (e.g. large plasmids with single or multiple inserts). Use a primer design program such as NEBuilder® to design cloning primers that anneal outside of any repeat elements.