plasmid isolation - (May/28/2009 )
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
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.