Problem growing colony in LB - (Jul/15/2005 )

Hey all,

I'm trying to ligate a 500 bp insert into pet28a vector. I think i've managed to get the ligation to work, but I only get one colony on my agar plates. When I try to grow that colony overnight in LB using the same antibiotic (kanamycin) it doesn't grow. Does anybody know what might be wrong?

Getting only one colony is dangerous.

Did u have proper controls during ligation reaction, like vector and insert control?

How about their results on transforamtion?

I also have the problem.

Usually, i think it is not the target colony, and abolish it.

check you antibiotic concentration and make sure it is appropriate
p..

I had such similar problem before (colony dosen't grow in LB). It was solved when I changed a new batch of plates, I don't know why, probably due to the antibiotics.

Old plates with a low concentration of antibiotics are always a problem.