Restriction digestion of PCR fragments

Andy Kravetz akravetz at Walden.MO.NET
Fri Aug 18 13:24:13 EST 1995

B VISSER * x2818 Plantkunde * (pbv at 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

Washington University School of Medicine
Biochemistry and Molecular Biophysics
St. Louis, MO
kravetz at OR
akravetz at

"I love the wild power of language and and the purity of the madness that
governs it and makes it music" 
                                        -HST, _Generation of Swine_

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