Loss of insert after cloning! - (Jul/24/2007 )

Hi,

I have a weird problem. I am trying to clone a gene into the insect baculovirus vector pFastbac1. Its directional cloning. The insert is generated with appropriate REs by PCR. Now what happens is that after transformation, I screen my colonies by colony PCR and I get positive transformants. When I scale up the positive colony and extract plasmid there is no insert - by PCR and RE digestion. This has happened twice. The insert is a stable one with no hairpins and other unstable structures. I use DH5a for this. I have never had this prob before. Is it possible that just amplified product or maybe vector-insert ligated at one end (and therefore linear) has got transformed in 1-2 cells and gets lost during cell division? But I don't understand how that 1-2 cells is picked up everytime in PCR.

Any explanations and suggestions?

Thank in advance!!!!

Are you 100% sure your colony PCR excludes colony's with plasmids that do not contain your insert?

ie did you have one primer binding to the insert and the second primer binding to the vector. Colony PCR is sensitive enough to pick up DNA inserts leftover from the ligation reaction. So if both primers bind to the insert, a signal will light up.

In reply to both the questions:

During the first expt. I screened about 8 colonies of which 5 were positive and 3 negative. PCR mixes were also clean. During the second time, I screened 10 and all were positive, PCR negative controls were clean. I also noticed that when I add a little more of colony (I pick it up with a tip and transfer first to a tube containing media and then dip it in the PCR mix) I get brighter (more intense) amplified products.