Restriction digestion of PCR fragments

[Christopher D. Skory]

I always add an extra "GCG" on the 5' ends of my primers when 
introducing an R.E. site.  After PCR, the fragment is cleaned up 
with Eluquick (S&S) and then digested.  In my experience, if it 
is unligatable (tested through self ligation or cloning) after 
this, then TA cloning (followed by R.E. digestion) is the only 
alternative.  I have never gotten a fragment cloned by using 
the extra cleanup steps with phenol, proteinase K, or whatever 
cleanup that pleases you.   The glass milk works great if the 
sequence will allow digestion.  Otherwise, you're stuck doing the 
extra step with TA cloning. 

Just my two cents worth.

Christopher D. Skory, Ph.D.  
Research Microbiologist

National Center for Agricultural          
  Utilization Research, USDA-ARS               
Fermentation Biochemistry Division
Peoria, Illinois 61571-3902                          

E-mail:  skorycd at ncaur1.ncaur.gov



In article <DDn3MF.279 at news.nsw.CSIRO.AU>, 
D.Adelson at prospect.anprod.csiro.au says...
>
>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.
>
>