TOPO® TA cloning - (Oct/22/2008 )

Hi all,

Recently I am having strange problem with TOPO® TA cloning (pCR®2.1 vector). My PCR is clean, I do the TOPO reaction and the transformation, I have many many white colonies. When I cut with the mini DNA wiht EcoRI, I see my PCR product ( insert) and 8-10kb band instead of 3.9kb topo 2.1 vector. Any idea?

Cheers

Burcu

Hi all,

Recently I am having strange problem with TOPO® TA cloning (pCR®2.1 vector). My PCR is clean, I do the TOPO reaction and the transformation, I have many many white colonies. When I cut with the mini DNA wiht EcoRI, I see my PCR product ( insert) and 8-10kb band instead of 3.9kb topo 2.1 vector. Any idea?

Cheers

Burcu

Thats the only thing I can think of. Concatemerization.

But technically you can't get concatemerisation can you? The vector is pre-cut with a 5' A overhang on the as strand right?. For it to swing around and bind you would need an alternate 3' T overhang therefore (but it's blunt)? - If it is the blunt end binding itself would be a lot less efficient (maybe try cutting down the amount of ligase).
Your reverse primer doesn't end in CACC does it? (the Topo TA recognition site for the start).