Restriction digestion of PCR fragments

Mike Dalrymple dalrymple at pplros.demon.co.uk
Fri Aug 18 10:53:20 EST 1995


In article <pbv.78 at rs.uovs.ac.za> B VISSER * x2818 Plantkunde *,
pbv at rs.uovs.ac.za writes:
>>Hi
>
>I am in the process of digesting amplified cDNA fragments with Not1 of which 
>a restriction site was incoporated into the primers.  I am not able to fully 
>digest the fragments, even though sufficient nucleotides are present on the 
>5' side of the restriction site.  Could Ethidium bromide play a role in the 
>inhibition of the digestion of the DNA?  I usually run the fragments on a 
>prep gel and cut the desired fragments from the gel with phenol/chloroform 
>which is followed by a ethanol precipitation.  Could this be the reason?
>
>Botma Visser


  Could be.  Why not phenol extract the PCR product, cut, then run the
gel?  I 
  suspect that would save you a gel step anyway.  Having said that, I
have often
  tried putting sites in the primer, and half the time the quickest way is
  to clone the PCR product into a T vector and then cut it out with the
required 
  enzymes. It's annoying, but there you go.

Anthony



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