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colonies not growing on plates, but in culture - (Feb/14/2008 )


I am hoping someone might have some suggestions on this:

I have a plasmid that I am trying to transform. I have CMAX 5alpha F (BioRad) and XL-10 Gold (Stratagene) competent cells. I have followed both the protocols to transform them, and I get growth during the 1 hour incubation at 37C. The problem arises when I try to plate them. I am using homemade LB plates with 50ug/ml and plates bought from Fisher that are LB + 50ug/ml. I plated 100ul and 200ul on to one of each plate. I incubated them at 37C overnight. No colonies grew. I have repeated this 3 times. When I use some of the original transformation stock into LB + Amp liquid stock they grow up really well. I have no clue what is going on, and would appreciate any help that I can get.



How much DNA do you use to transform? Verify the antibiotic resistance gene in the plasmid.