Problems cloning a tiny insert into a huge vector - (Oct/31/2007 )

I’m trying to clone a PCR-amplified 750 bp DNA fragment into a 14Kb vector for 4 months now and this has not been very successful so far...

Brifley, the strategy is:

I amplify by PCR the insert using primers that containing the extremities restriction sites for AscI (forward primer) and PmeI (reverse primer). Double digest the insert and the vector ON with the above-mentioned enzymes and they give clean and sharp bands of the expected sizes 750 bp and 14 Kb, respectively. Ligate insert and vector at a ratio of 7:1 and chemically transform E. coli DH5alpha.

I obtain around 30 colonies. However, when I extract DNA from these transformants and digest it with the same enzymes used to clone I have a pattern of bands that makes no sense to me. Two faint bands of around 7 and 8Kb each and another strong one of around 4 Kb.

Thanks in advance for any suggestions!

mariazeitona

What plasmid are you workin on, does it contain repetitive sequences (viral LTR's)? How big is the part you cut out your 14 kb vector? (it has to be large to be able to visualise single cut from double cut vector). How many extra bases do you add to your primers next to you restriction sites? Maybe first clone it into another vector (depending on your polymerase a blunt vector or one with overhangs) and then cut your fragment out (this way you're sure it's been cut out).

What I have done in the past is to first create a vector that has a part of the original vector (say 3 kb into a pUC or pBKS vector), clone your 750 bp into that one and then clone it back into your bigger vector.

Good luck!