cannot isolate plasmid from mutants - please HELP! (Aug/18/2005 )
hi. i've been trying to sequence plasmid from mutants of an integrase protein but they do not sequence. the mutation reaction was trasnformed into XL-1 blue cells, and the cells grow fine on the amp plates. they grow a little slowly in 1 ml cultures, but take a while to grow in 5 ml cultures. i've isolated plasmid from those cells several times using different kits, (Qiagen, Eppendorf, Templiphi from GE) but nothing sequences. i tried new sequencing primers, mutagenic primers, etc. i've changed plates, broths, even the xl-1 blu cells. any ideas as to what could be happening?
What do your plasmids look like?
Do they digest with appropriate enzymes and make right size bands? (ie confirm vector backbone)
can you drop the insert out and is it the right size? (ie confirm insert)
can you sequence from this plasmid with the non-mutant template?