help: primer restriction sites

sui sui
Mon Sep 4 23:15:51 EST 1995


In article <424pki$rhl at grandcanyon.binc.net>, 
movatech at mailbag.com says...
>
>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


There is a table about this problem in the New England Biolabs 
Catalog, which lists how much in % of the enzymatic efficiency 
you still have with n additional bases...

Sui Huang
Children's Hospital, Boston




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