Cloning large pcr product - efficiency (Jul/21/2005 )
I tried to clone in TOPO TA plasmid 1 kb PCR product, extracted from the agarose gel, but there was extremely low quantity of colonies. I increased time of ligation reaction and added more of PCR product, but results were similar. May be You know how to improve transfection efficiency in this case?
first did you test your colonies for positive ones?
for cloning in topo TA follows rules of invitrogen and it works well. But i prefer a day ligation than an overnight ligation.
If you are using the TA TOPO kit you might have chosen primers that don't allow the 3' A overhangs to be added.
I have also found that the more you do to fragment (like gel purification) the few colonies you get from TA cloning. I don't know why this is, but I have always assumed that the 3' A overhangs are easily damaged. Can you try cloning without gel purification?