Currently having trouble cloning some PCR cloned fragments (digested with NheI and XhoI) into a pGL4 vector.
standard mix is 100ng of digested and TSAP treated vector and 3x molar amount of insert (also RE digested) and using NEB quick ligase for 15 mins at 25 degrees.
I get absolutely zero colonies on vector alone plates (positive control plate has many many colonies), while the addition of my insert only yields fewer than 8 colonies and these all seem to be religated vector.
Any tips?