Restriction digestion of PCR fragments

David Adelson D.Adelson at prospect.anprod.csiro.au
Sun Aug 20 21:44:39 EST 1995


In message <412lsd$3s at Twain.MO.NET> - akravetz at Walden.MO.NET (Andy Kravetz) 
writes:
>B VISSER * x2818 Plantkunde * (pbv at rs.uovs.ac.za) wrote:
>: 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?
>
>I am having the same type of problem as you. I am trying to cut the ends 
>of a 400bp fragment amplified so I can use it in a ligation. It appears, 
>dispite having extra bases that it is not working well. I am considering 
>running it on LMP and then just cutting it in the agarose, then doing the 
>ligation in the agarose. If I do this, will I have any problems and can 
>anyone else see a problem with this. Thanks
>


This topic came up in a thread about six-twelve months ago.  One suggestion 
involved proteinase K digestion of PCR products prior to restriction 
digestion.  I tried it and it cured my restriction digestion problem 
brilliantly.  The reference is:
  

  Crowe et al (1991) NARS Vol 19 p184.

Hope this helps.

******************************************************
*Dave Adelson      CSIRO Divison of Animal Production*
*"What, me worry?"    Sydney Lab, Sydney, AUSTRALIA  *
*ALL OPINONS ARE MY OWN, USUAL DISCLAIMER APPLIES    *
******************************************************




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