problems in subcloning PCR products for TA cloning - (Apr/09/2006 )

Hi everyone,

I want to subclone small DNA fragments (range from 200bp-1kb) into TA cloning pGEM-T vector. However, too many white E. coli colonies were recovered after electroporation, but none of them contain the vector after mini-preparation (i.e. NO vector inside the cells!!!). I have tested the efficiency of the drug by spreading non-electroporated cells on those agar plates. But I found that the drugs were active. So have anyone of you encountered similar situation before or can anyone of you think of any possible solution?

THANK YOU VERY MUCH!!!

The antibiotic is effective against your host strain, but not against your contaminant -- something you're using is contaminated.

What is your host strain?

Why are you using electroporation? Can you not transform your cells if they're chemically competent?

Really thank you for your reply!! Can you think of any source of contamination? Is this due to the ligation mix, broth or contamination in my host strain?

My host strain is XL1-Blue. I usually use electroporation due to it high transformation efficiency than heat shock those cells.

THANKS ALOT!!!!

Well, it's probably not your broth, or else you'd see it, but it could be nearly anything else (pipette tips, pipettor, electroporation cuvette, dH2O, anything you use to make your cells electrocompetent, etc.).

If you think your stock cells are contaminated, streak them for single colony isolates, and pick a single colony (this was why I asked what E. coli strain you were using -- XL Blue has Tn10, and thus is tetracycline resistant. You can select a colony on tetracycline plates, or try your transformation on plates that contain tetracycline and whatever antibiotic marker your vector carries).

TA cloning is very effcient, as is transformation of chemically competent cells. Were I you, I would do it this way, and forget about electroporation, which isn't necessary for a routine cloning, especially of such a small vector+insert clone.

this might be very obvious - but your concentrations of IPTG and XGAL are right yeh?