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cells grow but low/no plasmid yield


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#1 sharmistha

sharmistha

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Posted 01 October 2010 - 08:01 AM

Hello,

I'm trying to grow a plasmid on a PCMV6M backbone which I've successfully done in the past. Recently my glycerol stock has not been giving me good growth so I retransformed my plasmid, plated it and tried growing it. At first my cells were either not growing at all or growing poorly overnight so I would keep growing them for an additional 5hours until they reached a good density. However, each time that I extracted the plasmid using a mini or midi prep kit I am getting an extremely low yield (30ug/ml for mini and the best i got for midi is 100ug/ml). I have tried transforming the plasmid in DH5a, JM109, XL1-Blue, and BL21 and whenever I get good growth I don't get plasmid. I've also transformed different older plasmid preps we have in the lab as well as plasmid stocks from other labs. I've used carb and amp, i've tried LB and superbroth. I can't think of anything else I can try!! Please help! :blink:

Thank you!! :)




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