Mutagenesis PCR transforming into BL21(DE3) - (Nov/12/2007 )
I have performed a point mutation in a plasmid by following the Quick change protocol. After DpnI digestion of the PCR reaction is there any reason why I cannot transform this directly into a BL21(DE3) expression strain and inoculate a starter culture from the colonies I hopefully will obtain?
The transformation efficiency of BL21 strains is dramatically (1000x) lower than cloning strains. You want to clone into a cloning strain first, isolate boat-loads of plasmid with a miniprep, then transform into BL21. This also has the bonus of letting you sequence the plasmid and keeping it in the freezer with much higher quality than you can get from a BL21 miniprep.
BL21 strains are usually used for protein expression. There might be other cells that you could try for cloning.