Digestion at the termini of PCR product?

davesmith at bioch.tamu.edu davesmith at bioch.tamu.edu
Tue Nov 28 16:54:43 EST 1995


imazhq at leonis.nus.sg (Zheng Huanquan) wrote:

>I currently get a PCR product which has Nhe I at 5' terminus and BamH I 
>at 3'. I would like to ligate it into pET21a. Any one has experience in 
>digesting DNA with enzymes which sites are at termini? any protocol?

>Thanks!


>H Zheng


I've never used NheI, but I frequently use NdeI (a sorry enzyme!) in
conjuction with BamHI to sublcone PCR frags into pET11a.  In our hands
we have discovered that NdeI is an "endophobic" restriction enzyme.  I
see from looking at the recent '95 New England Biolabs catalog on page
208 that NheI is also "endophobic"--meaning that it does not like to
cut near the ends of DNA fragments.  Although the extent of the
termini that NEB put at the end beyond the restriction site is
limited, you can get the picture of what extending the PCR product by
a few base pairs can do for you.  We have basically eliminated the
problem by following their example for NdeI.  We ordered a new primer
which encodes an EcoRI site and 2 extra base pairs 5' of the NdeI
site.  PCR products using this primer are apparently much easier to
clone--and when they DO give us trouble, we fall back to indirect
cloning by first cloning the PCR product into, say, pUC19, or some
other vector by EcoRI/ BamHI, etc.  EcoRI does not seem to care how
close to the end of DNA fragment the site is--it's a very healthy
enzyme!
Hope this works for you!
dave smith




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