Hello all. So, we transformed RbCl E. coli DH5alpha cells using 50ng of DNA. But, we did not get a single colony. Can anyone explain the reason behind this problem?
Note: RbCl comp cells, Ampicillin stock and LB+Amp plates were freshly made.
What controls did you do? If you got no colonies then the likely explanations are either 1) you don't have plasmid DNA (so use a different plasmid stock as a control; or 2) your competent cells were not any good (try your plasmid stock in different batch of competent cells); or 3) something was wrong with your plates (again try a control). Once you know which is the problem you can begin to troubleshoot.
Thanks Michael.
Possibility 1 could be the reason
Possibility 2 - competent cells were good cause I got colonies for other plasmids
Possibility 3 - Plates were also good
Control 1: Cells without plasmid in antibiotic containing media plate and there was no growth
Control 2: Cells without plasmid in plain LB agar plate
Sounds like the issue is the plasmid DNA then. You could try to transform it into some different cells to see if it works, or look at the DNA on a gel to see if you have DNA.
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