4 replies to this topic

[biochemistry23]

Hi everyone!!!!

I am trying to put a PCR product  (850bp) in the vector pet20b. I am doing the ligation at 16 degrees O/N and then I transform DH5A competent cells and plate them in plates with LB and ampicillin. Finally, I get no colonies or just one colony without the insert.
Do you have any suggestions? Thanks a lot!!!!



P.S. the self-ligation works correctly.

[San Diego Mike]

Just to clarify your question: when you self-ligate your vector and transform DH5alpha, you get colonies, but when you clone in your PCR product and transform, you get no colonies? If that is the case, it sounds like there is something in the ligated vector that is preventing transformation.

[scrat]

Do you have restriction enzyme sites within your PCR primers? If so, it's hard to check whether the digest has worked, how far are the restriction sites from the end of the product sequence? If you are really stuck, TA clone the PCR product into a nice easy TA plasmid, check the sequence, then subclone into pet20b. Time consuming but more likely to work.

[biochemistry23]

San Diego Mike, on Apr 22 2010, 11:29 PM, said:

Just to clarify your question: when you self-ligate your vector and transform DH5alpha, you get colonies, but when you clone in your PCR product and transform, you get no colonies? If that is the case, it sounds like there is something in the ligated vector that is preventing transformation.

yes, that's exactly the problem! any suggestions or options????

[San Diego Mike]

I think Scrat has you covered there.  I agree with him.