FAQ: What guidelines are recommended for improving clone stability?
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.