Restriction digestion of PCR fragments

[Andy Kravetz]

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

Andy
Washington University School of Medicine
Biochemistry and Molecular Biophysics
St. Louis, MO
kravetz at dciris1.wustl.edu OR
akravetz at waldeno.mo.net
http://mbb.wustl.edu/~kravetza/index.html
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