Adding restriction sites to PCR primers

Chris LaRosa clarosa at biocomp.unl.edu
Tue May 16 09:13:15 EST 2000



 do
> I need to add extra bases after the enzyme's site so the the enzyme will be
> able to cleave it?
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The preferred approach is to add the restriction site and extra bases,
the number depending on what site your are adding.   EcoRI site
according to charts in the back of NEB catalog say that 1 extra base is
sufficient, but some sites require may more.   As a rule of thumb I
always chose 5 extra bases.   
I suppose one could add just the half-site, but you will need to by
kinased primers or kinase the pcr product after production.   Perhaps
some  more expert experts can comment on pcr cloning.   BTW there are
sites on the web that go into these
details...//res2.agr.ca/winnipeg/cloning.htm
www.nwfsc.noaa.gov/protocols/pcr-cloning.html
wint1.sb.fsu.edu/bch5425/lect24/lect24.htm




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