Hello everyone,I am in the need for desperate help after failing to clone a 2.3 kb insert to a 3.5kb target vector for a few months now.
This is what I have done so far:
1. Run a PCR on the gene from a library (on a plasmid source)
primers with restriction sites for XbaI and SpeI corporated.
2. digestion with the restriction enzymes of the very nice PCR product, and ligation to the target vector. This ligation (ratios 1:3 1:6 1:10 vector:insert) did not work no matter what I did (with and without CIP, and with a sequential cut of the vector). The target vector was verified to be linearlized
3. Assuming the PCR product does not cut efficiently I subcloned it into an intermediate vector Next, and cut out (step-wise) the insert. Now I was sure that the insert is cut at both ends and has the right overhanging sequences for insertion to the vector. But again, the ligation did not work (different T4 ligases, different ratios,different compatents). The target vector was also verified to be cut with both of the enzymes.
4. desperately I now ordered new primers with different restriction sites.
Most of the time I get no colonies at all and sometimes I do get some colonies both on the plate with only the target vector as well as the one with the ligation mixture but none of those is positive for my insert.
Can anybody offer me a piece of advice on how to proceed?