TOPO cloning problem - (May/13/2009 )

I did TOPO cloning yesterday and spread the bacteria onto LB ampicillin 50 ug/ml and culture at 37 over night.
BUT NOTHING grows out!!!!

TOPO vector 1ul
PCR product 4ul
salt solution 1ul
total 6 ul, room temperature let react for 30 min.

then I did the normal transformation into top10 cell.
and I did one positive control--pUC19.

and grow them onto LB plate containing ampicillin 50ug/ml.
culture plates at 37 over night.

Nothing on the TOPO vector & PCR product.
pUC19 plate grow fine.there are lots of colonies on pUC19 plate.

WHY?

What polymerase did you use? If you used a proof reading one, they have an exonuclease capability that means that they don't leave A overhangs on the end of the PCR product - something Topo TA cloning relies on.

I second bob1. In case if you have used proof reading polymerase, there is a protocol in TOPO manual on how to add -A overhang. It should work after that.
If your workflow was normal and still you had no colonies, don't ask why, you don't know what bugs do, just repeat, you may get it.

I am not a real fan of TOPO, I just prefer cut and paste, thats more reliable than TOPO. It takes time but its worth it.

did u extend the final extension time to like 10 min. that 's required to add the extra A tail.

But even if gene of interest didn't combind TOPO vector,the empty TOPO vector itself should combind itself and be transformed into TOP10 cell.Since TOPO vector has the amp resistance,they should grow on the LBamp100 plate after culturing over night.

Since according to lots of people's experience,you should see lots of colonies over night in TOPO cloning.

travelhappily on May 13 2009, 04:53 PM said:

I've never used TOPO so not sure of protocol, but... arent you mising the Ligase in this reaction???

almost a doctor on May 21 2009, 08:01 AM said:

the ligase used in this experiment is called topoisomerase which has already existed in the vector solution.

travelhappily on May 21 2009, 07:47 AM said:

Actually, no - topo cloning precludes this possibilty, you should not get empty vector clones as the presence of the topoisomerases added to the ends of the vector cause steric hindrance, preventing self-ligation.

travelhappily on May 21 2009, 05:50 PM said:

Didn't know that, cool!

In that case I do agree with bob1 and co. make sure you A-tail your PCR product. If you are using a proofreading polymerase, all you have to do at the end of your PCR is add extra dATP and Taq and icubate at 72C for 15-20min.

Good luck!

almost a doctor on May 22 2009, 01:12 AM said:

Thanks so much!!!
I will try to elongate for 10min since I used 5 min in elongation before.

By the way,how carefully should I treat TOP10 cell to make sure the efficiency of the cells?
They are so ......weak!!!!!!! Make me crazy!!!1