funny result from pGEX-insert cloned in DH5 alpha - insert is the wrong size! (Oct/25/2008 )

Hi!

This has happened with 2 different constructs, however the insert is the same size for both. I digested my PCR product (600 bp) and pGEX vector (4.9 kb) with BamH1 and EcoR1, then gel purified them. They ran at the right size. Ligated, transformed in DH5 alpha. Uncut vector grew a lot of colonies, digested vector grew none, suggesting that both restriction enzymes cut. pGEX + insert ligation grew colonies. grew many of these colonies overnight, minipreped, digested, and ran a diagnostic gel.

almost all samples (17 total) had cut pGEX and some uncut pGEX, as well as bands at about 900 bp for one construct, and 800 for the other... while my insert for both constructs are 600. There were no bands at 600. Most also had bands between 2-3 kb.

My PCR shouldn't be the problem since my gel purified product ran at the right size. I also did PCR cleanup. The pGEX seems to also have been digested ok, and the pGEX and DH5 alpha have worked fine for other people in the lab. should be no star activity since previous digestions showed none.

what in the world could the 800-900 bp fragments and the 2-3 kb fragments be?? The only ones i should get are 4900 for cut pGEX, a little lower for uncut pGEX, and 600 for my insert. And even if there was contamination following the transformation, where is my insert? I will probably get them sequenced if I can't figure it out..

has anyone else had this problem? something funny must have happened in between my transformation and digestion. maybe its because halloween is coming up. oOOoOOooooO.

thanks!

You have evidence that your vector cut properly, but none that your PCR product was cut properly, other than the fact that you recovered no bands of 600, which suggests it wasn't. All kinds of strange insert sizes come up in all cloning experiments, but if your insert is correct, at least a few should contain it.

Is the BamHI and EcoRI fragment an internal segment of your PCR product, or did you engineer restriction sites onto the 5' ends of your PCR primers? If the latter, how many irrelevant bases did you include 5' of the restriction sites on these primers?

The other (rather remote) possibility is that your PCR product did cut correctly, but the correct pGEX clones are lethal to E. coli. Is this a possibility?