PCR cloning - PAGE

Steven Enkemann enkemans at dc37a.nci.nih.gov
Thu Aug 31 18:32:01 EST 1995


In article <1995Aug22.215624.59506 at cc.usu.edu>, jmorrey at cc.usu.edu (J.
Morrey) wrote:

> We have tried unsuccessfully to clone 3.0 and 4.4 kb PCR products into
> plasmids.  We have employeed many different strategies including
> blunt-end ligation after Klenow fill-in and kinase reactions,
> fractionation of the PCR product by agarose followed by restriction
> cutting and ligation into the vector, blunt-end ligation after agarose
> fractionation, etc.  We have empirically observed that agarose-purified
> PCR product (via agarase enzyme) may be inhibited by perhaps
> contaminants, thereby yielding no cloned DNA.  Consequently, we are
> considering fractionation by PAGE and subsequenct purification by
> elution.  Here are the questions...
> 
> Will polyacrylamide gel electrophoresis likely yield less inhibitory
> contaminants which would lead to cloned PCR products?  Will 3.0 or 4.4
> Kb fragments elute from the polyacrylamide during the purification
> process?

   Two questions.  Do you know the sequence of the products you are trying
to clone?  Or at least a restriction map.  And do you know that your
problem is in ligation and not in the growth of the resultant clones?
   The reason I asked the second question is because we had trouble a few
years ago with a fragment that contained a poisen sequence so the clones
grew very poorly.  The reason for the first question is for possible
solutions.  When I first tried to clone large PCR fragments I had a lot of
trouble.  But I could cut my PCR product in half with a restriction digest
and clone it in two pieces with one blunt end and one sticky end.  I found
the poisen sequence because only one of the two halves would clone. 
Cloning the poisen sequence required some further tricks but this method
might be informative as to where your problem lies.
   Many people have posted that Proteinase K treatment is very helpful. 
This is absolutely true.  I routinely treat my finished PCR reaction with
30 minutes of klenow and then Proteinase K as described by Crowe et al.
then P:CHL extract and blunt end clone.  We usually do this into a Sma I
site.  The Sma I site is destroyed upon ligation so background can be
eliminated by digesting with Sma I before transformation.  The enzyme Sma
I works well in ligation buffer and at low temperatures so it can be added
right to the ligation.  The only problem is if your fragment has a SmaI
site.  One last comment.  I useally don't let my last PCR elongation go
for an extended period of time as many protocols recommend.  No data but I
believe that this is where the extra A comes from.

Steven A. Enkemann PhD.

I don't believe that curiosity killed the cat.  
I think it just did the wrong experiment.



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