Hi,
I'm facing a recurent problem with the cloning of a given cDNA. Everytime I'm doing the cloning I end up with a mix of big and small colonies on the insert+vector plate while getting only few big ones on the vector alone plate. The small ones are clearly not satellites, I know how do they look. I picked some of the small one but they are generally not growing in liquid. Tried to grow plates at 30°C no changes. Ran my ligation on gels and they look very efficient. I clearly don't know what to do. Any suggestions? Thanks
Are you cooling your agar sufficiently before adding the antibiotics? Have you tried doubling the antibiotic dose?
Plates are pourred by our local facility and did not get any problem with them so far.
Can you please tell me how much of transformed cells you are spreading onto the plate
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