problems with restriction digestion of plasmid DNA

Vladimir Svetlov svetlov at oncology.wisc.edu
Tue Nov 18 15:19:33 EST 1997


In article <64s031$1vbk$1 at www.univie.ac.at>, harald at mol.univie.ac.at wrote:


 > 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). 

Is there an EagI site in pGEM-T? None of the sites you've listed are on the
map in the Promega catalog - if you are looking for an insert, why not use
double digest with smth. like SphI+PstI or ApaI+SacI?

> 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?

This last sentence rules. I don't know about everyone out there but I
really have not caused your endonuclease digest problems <G>. 

> 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? 

Depending on how you've done the prep contamination can be of protein, RNA
or carbohydrate nature. Phenol/chloroform extraction, using smth. like
Promega's PCR clean-up kit and even simple high-salt ethanol precipitation
would help to some extend. Chloroform extraction is often essential for
downstream processing of the DNA since even traces of phenol can inhibit
many proteins. Another reason for your plasmid preps being refractory to
the nuclease digest could be an excess of EDTA or other LMW chelators. A
10-fold increase in EDTA conc. in the prep would not affect appearance of
the DNA on the gel but would compromise most Mg and Zn dependent nucleases.


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

If the contamination is a problem (and not the absence of sites in the
plasmid...), you can most reliably salvage your preps using PCR clean-up
kit. Use any reputable kit for re-prepping (e.g. Quigen, Promega, or
Pharmacia (the latter for the endA- host)). If you are opting for the low
cost option which gives a very nice purity, try you usual alkaline lysis
prep in conjunction with the BIO101 Miniprep Express Matrix (the addition
of matrix increases the cost of your prep by about 10 cents - compared to
0.5 -1$ per prep for an out of the box kit). 

Regards,
V.

-- 
Vladimir Svetlov
McArdle Lab for Cancer Research
Dept. Oncology
UW-Madison
1400 University Ave.
Madison, WI 53706



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