Confused ligation - (Apr/16/2001 )
My amp plates are always flooded with satellite colonies even within a period of 16-18 hours of incubation. What do I do? Is there any other cheaper analog of carbencillin? I have tried spreading the amp also to counter heat inactivation while pouring- doesnt seem to help. help!
i have same experience in the past, you can try to use a fresh make Amp plates i.e.use the plates which make in the same day.The problem will be less serious.
What's the Conc. you are using? You may use 100ug/ml. Another suggestion is to short the incubation time.
Use lower insert to vector ratio (1:1 to 3:1) and lower DNA concentration. If protein is toxic you might need to use another vector.
Clontech sells a PCR random mutagenesis kit.