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plasmid isolation - (May/28/2009 )

hi all

Im trying to isolate a construct which i transformed into E.coli cells .The construct has vector backbone of pUC8 which is a high copy number plasmid but im nt able to isolate plasmid at good concentration for further work. How can high copy number plasmid come as 50ng/ul concentration? I keep the bacterial culture in 37C overnight condition the growth seems to be okay but very less concentration of plasmid

Any suggestions?


How are you isolating your plasmid?
Did you isolate a control plasmid to see if that works fine?


need more info than what you have provided.

what type of plasmid prep are you perfoming? alk. lysis? your own sol'n's? a kit? manufacturer?

from the limited info you provided it sounds like incomplete lysis or sol'n 2 has too much NaOH such that sol'n 3 does not neutralize plasmid or your wash buffer (if column prep used) hasn't the right amount of EtOH added or your elution buffer pH is off etc. etc. etc.

a 3mL o/n culture of puc dna using a qiaprep column with a 50uL elution should give you btwn 400 - 800 ng/uL of plasmid measured by nanodrop spec.


For example, you should use N3 instead of P3 for spin-column miniprep. But you got very low yield of DNA just because you used P3.

-Functional Screens-

Insert will affect the copy number of ur plasmid. the bigger it is the lower ur end copy number will be. don't worry about this . 50ng/ul is not too bad.
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