cloning 708bp PCR prodyct into pET101 vector (TOPO) - (Oct/15/2007 )
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.
I'm also having trouble with getting the insert into the plasmid... Maybe something from these replies may help?
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