insert is being kicked out during cell growth - (Jan/21/2006 )

hi all,

I am trying to clone a 6.5 kb DNA into a expression vector. The insert gets ligated and I get some colonies on the plate. When I screen by PCR for the insert I get a PCR product of the right size. However when I grow the colonies in LB and antibiotic and isolate the plasmid I loose the insert (I checked the purified plasmids by PCR). Can anyone help me with this problem?

Thanks much,

Rohit

are you quite certain your primers have no sequence homology to your host strain?

have you checked uncut plasmid prep on a gel, too, to be sure you are getting plasmid with your prep?

this is very puzzling...

I have checked for the primers and they have no homology in the strain.

Thanks for the reply

doing PCR on colony's is an easy technique, but it generates a lot of false positives somehow. Half a year ago, I did colony PCR on 40 colony's of ligation of cut PCR + cut vector and also a colony PCR on empty vector self ligation and ALL of them were positive... After purifying just half of them were positive by restriction digest...

You indeed need to check the size of your plasmid on gel, both uncut and single cut.

Apart from this, shed some light on your cloning strategu + how many colony's do you generate on each plate (PCR + Vector, empty vector).