Topo cloning no colonies - (May/19/2005 )

I am trying to clone and sequence HIV envelope from proviral DNA in patient PBMC. I have been using Platinum taq and nested pcr to get my product (which I get) and gel purify the band before ligating. I am ligating with the Topo TA kit and when I check the ligation I am getting some ligated product, some empty vector and some unreacted pcr product. I tried adding TA overhangs but still got some empty vector. When I do the transformation I get very few or no colonies on Ampicillin plates. I'm running out of ideas- help!

how old are your competent cells??

it could very well be that they are not so competent after all

Nick

Agree with Nick.

To test if your competent cells are right, transform your cells with intact plasmid such as pU18 usually included in TA cloning kit. If the cells are ok, you should definitely see tons of colonies.

It seems that cell viability was the problem. Thanks guys.