TOPO cloning problems!

[annabcn]

Hello everyone!
I'm new in this forum, and I hope you can help me with my big big problems with TOPO cloning!

I'm using the pcDNA3.1TOPO vector from Invitrogen, and when I do all the protocol, my results are horrible!!

What I do is:
1) PCR of my insert with EcoTaq
2) gel purification (cut the band of my amplification product and purificate the DNA with the Promega kit that consists in a column purification).
3) Mesure the DNA concentation to ensure that I have recovered my insert.
4) Perform the cloning reaction: 4 ul of DNA, 1 ul of TOPO and 1 ul of salt solution.
5) incubate the reaction 15 minutes at room temperature
6)transform the reaction in OneShot competent cells by chemical transformation.
7) spread the cells in LB+Ampi plates.

Then, I find some white colonies (15-20 or more), and when I analize them by restriction to check my cloning products, I find that all of them contain the TOPO vector empty, without my insert!!!

So, how can it be possible?? How can the TOPO vector recircularize and don't incorporate my insert???

What am I doing wrong????

PLEASE HELP ME!!
If you have a better protocol to clone with this vector, or there's another commercial vector that gives better results, please tell me!

Thank you very much!

Anna

[admin]

Hi Anna,
I think your main issue is with your PCR and your purification of it. Have you checked your PCR on a gel? Is there only 1 main band? Is there any primer dimers showing up on the bottom?

I would cut and purify the main pcr band and use that for ligation immediately (ie fresh).

I have done many Topo clonings and never had a problem, but I would mostly do a good PCR with one band first and if there were many bands I would always purify the one I wanted to clonel.

cheers