question on TOPO TA problems - no positive colonies - (Mar/05/2006 )

I tried twice to clone a 1.7kb pcr product to a topo 2.1 vector. I dont use X-gal, so I just pick colonies and check them. I checked 10 colonies each time and none of them was positive for my insert. Is that normal? Would you suggest that I pick more colonies, or I should go back and try again after changing the conditions of the reaction?
Thank you all

you may change the antibiotic on plate. Topo are holder of amp and kana resistance genes....
you may in second increase ratio of insert and re add poly A tail?

Thank you for the reply, although I am still a little confused. I can understand the change of the ratio, but what is the polyA tail you are reffering to? And how can the change of antibiotic make any difference?

Thank you again, I appreciate your help

Taq polymerase adds an extra A at the end of your PCR-product, that's the tail fred's referring to. Topo-TA cloning is based on the fact that your PCR products has this extra A, so the vector has a T-overhang that is "activated" with topo-isomerase so it can bind with the A and form a circular plasmid (if this even occurs on both sides with the same PCR product). After some time though, this extra A gets off your PCR product, so you might want to incubate your PCR product with fresh taq and dATP for 10-30 minutes @ 72°C to make sure your extra A is there.

vairus is correct. You have the T and A complimentarity for that purpose. There is also a topo version without topoisomerase linked. But works too.

Generally, PCR is done with a plasmid as a template. So the plasmid holds in majority of cases the Amp R gene. That's why by selecting on amp plates, you raise the topo AND the template plasmid. And as the plasmid template is good regarding quality, you find in majority on your plate the original plasmid. (to check, do an electroporation with 1ng of plasmid and you'll get the plate full).
But with kana, you select only topo vectors