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plasmid isolation


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4 replies to this topic

#1 deespike

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Posted 28 May 2009 - 09:01 AM

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?

#2 mastermi

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Posted 28 May 2009 - 09:15 AM

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

#3 eldon

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Posted 28 May 2009 - 09:27 AM

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.

Edited by eldon, 28 May 2009 - 09:30 AM.


#4 Functional Screens

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Posted 28 May 2009 - 06:48 PM

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.

#5 hanming86

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Posted 28 May 2009 - 08:23 PM

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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