I consistently get low yields with pAc5.1 constructs. I have tried growing cultures from fresh plates and glycerol stocks. I have also tried to use starter cultures (grown for 8 h) and I always get 3 to 4 fold less yields than the UAS ones have very high yields. I am considering growing them in terrific broth as opposed to LB. Any suggestions?
Hi Indrayani,
Whenever we are planning to do plasmid prep, we usually incubate the cultures in 200rpm shaking incubator at appropriate temp for atleast 12 hours to get very good prep. also i will suggest to grow your cultures with antibiotic pressure.
I hope it will help.
I would indeed incubate overnight. pAC5.1 has a PUC origin so it isa high copy number plasmid. Incubate as Anand suggested with ampicilline. Are you cloning (very) large inserts? Or inserts with inverted repeats?, both might give problems of stability. A very old trick is to add chloramphenicol 30 minutes before harvesting the cells. Upon chloramphenicol addition growth stops, yet DNA multiplication is continued. This used to be done in the pre-kit erra (when dinosaurs like me were still in the lab) to get higher yields.
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