cloning 708bp PCR prodyct into pET101 vector (TOPO) - (Oct/15/2007 )

Hi ,

I'm trying to clone a 708bp insert into the pET101 vector. I am using the TOPO kit (Invitrogen). When I perform transformation I have noticed I only get 2 or 3 colonies and none contain my insert. I have done everything the kits asks , my pcr is fine and so are my plates.

I am also using the company made TOPO one shot (DH5-alpha) competent cells.

I have also tried using another TOPO kit as well but still can't get my insert.

Thank-you.

I'm also having trouble with getting the insert into the plasmid... Maybe something from these replies may help?

http://www.protocol-online.org/forums/inde...showtopic=31073

well are you sure of polyA tail of your insert ?
If you do a purification, what is the 260/230 ratio value ? ethanol may inhibit topoisomerase