problems with restriction digestion of plasmid DNA

haral at mol.univie.ac.at haral at mol.univie.ac.at
Tue Nov 18 15:57:26 EST 1997


Hi, netters!

I´ve subcloned a purified PCR reaction product (about 323 bp) in pGEM-T 
vector, electrotransformated E. coli with this ligation product
and made several minipreps: some essentially as alkaline lysis with phenol 
extraction, others as in the Current Protocols in Molecular Biology 
(essentially alkaline lysis as well, but without phenol extraction step). 
However, these preps were not cut by restriction enzymes (I´ve tried EagI, 
ApoI and DrdI). Are there any of you what might be the cause for this? I 
strongly think that there are contaminations in the preps, but of which 
chemical kind and how to get rid of them? Some guys recommended me an 
additional phenol extraction step, others are more fond of reprecipitation 
from ethanol, still others prefer gel permeation. A different approach 
favored by some is using enzymes which are more or less resistant against 
common inpurities arising from these preps. Are there some candidate 
enzymes you could recommend? 

Any suggestions how to rescue these preps or at least for re-prepping are 
kindly appreciated!

Harald




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