No colony after ligation - (May/03/2006 )
I moved into a new lab weeks ago, I tried to construct vectors but no colony at all(even no background colony) after the ligation. So I tried to do self-ligation but also failed(single enzyme cut, recover from gel and ligate ON at 16C). My competent cells can be transformed by plasmids and TA vector(ligation-free), but not the ligation. I thought it shoud be the problem of ligation. I run a gel to check the ligation products and it looks good. I tried different ligase from 2 to company(invitrogen and NEB), they were same.
I've done a lot vector before, I don't know what's wrong I made in the new lab. Any suggestion would be appreciated.
what kind of efficiency are you getting with your transformation controls? supercoiled plasmid transforms at some crazy high efficiency....perhaps your ligation product is just transforming at a much lower efficiency and that is why it's not working.
Thanks for your reply.I use electroporation competent cells and one-tenth of my transformed cells can get more than 1000 colonies.I think it is enough for the ligation transformation. By the way, the colonies grow slower than normal ones, but I can get the plasmid from them. ......??
how much slower? perhaps there is too much antibiotic in your media?
20-24-hrs growths is as same as the normal O.N gowth(13-15). For Kan, I use 50ug/ml.
that sounds OK, although I think you can use a little less, 50 should still be all right
are you dialyzing or otherwise removing impurities from your ligation mix prior to electroporation?
are you dialyzing or otherwise removing impurities from your ligation mix prior to electroporation?
Thanks, I inactive the ligase in 65C and treat by chloroform before electroporation, this is the standard procedure I followed each time. I also did a control using the plasmid.