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Low yields of plasmids from miniprep - (Feb/12/2011 )

Hi all,

Recently I met a problem that the DNA yield of miniprep from the cryostock was extremely low. Previously it was fine when I picked them from fresh agar plates. I think there was something wrong after I stored the colonies at -80℃ with 20% sterile glycerol. The bacteria grows well after overnight inoculation, but it seems that the plasmids seldom replicates, whose final concentration is below 100ng/ul. I repeated it for many times and stricly followed the protocol, but the result didn't change too much. Can anyone give me a suggestion for this?



Are you sure the growth is done in an antibiotic containing medium? Loss of plasmid is common unless there is selection with an antibiotic.