No colonies with TOPO vector - (Aug/07/2007 )
I performed the TOPO cloning reaction and transformed chemically competent Mach1 cells and plated on Kanamycin plates. My PCR fragment is 850 bp. I did not get any colonies whatsoever, not even any background colonies. I know it is not a problem with the cells or the plates because I did a control reaction using a TOPO plasmid obtained from a colleague and obtained colonies with that. I ordered a new TOPO kit but did not obtain results even after using the new kit.
Can anybody suggest anything please?
mm well the recovery time for ligations which contain kanaplasmid is 1h at least.
then how did you prepare the PCR product ? regular taq or proofreading enzyme ?
i know that problems in my ligation with kana plates were that kana was too much concentrated. I reduced it by half when doing transformations.
Make sure that your PCR product has a 3' A overhang.
Secondly, perhaps your PCR product need to be fresh. Meaning, you need to do your PCR, purify, set up ligation, and transformation it immediately. This is what the senior in my lab is doing routinely. She found that she couldn't get the ligated product if she left her purified product overnight.
All the best to your experiment.
apart from the other suggestions:
How did you purify your PCR product (which has to be as fresh as possible)? Regular clean up or gel extraction with UV-exposure (this last one is really detrimental for topo, we switched to UV-free gel extraction and were a lot more succesful).