help: primer restriction sites

Jennifer Lyon movatech at mailbag.com
Thu Aug 31 11:56:50 EST 1995


Frank Chen <yatsen at wam.umd.edu> wrote:
> 	I designed a pair of primers which have restriction sites at 
> their 5' ends.  I did not add additional bp since I intended to clone 
> that into a PCR vector first.  I am wondering if I can directly digest 
> them with the enzymes for cloning into an expression vector.  I would 
> appreciate your help if you have done that for these two enzymes.
> 	The enzymes are: Bam HI and Hind III.


Basically, all enzymes seem to require at least some space between 
their site and the end of a PCR product for efficient cutting. BamH I
is one of the best cutters, but it still needs 3-6 base pairs. I've 
heard that in some cases, three is enough, but I'd recommend giving 
it 5-6 if you can. I would seriously doubt that you would get any 
digestion if the site is right at the end of the product without any
further space.

-Jennifer Lyon
Novagen, Inc.
JLyon at Novagen.com
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