help: primer restriction sites

[Rebecca Schall]

 Frank Chen <yatsen at wam.umd.edu> wrote:
> Dear Colleagues,
> 	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.
> Frank Chen
> 
Frank-
The NEB catalog is an excellent resource for enzyme activities (no 
affiliation with NEB!).  On page 209 of the 1995 catalog there is table 
of cutting efficiencies of a variety of enzymes close to the ends of 
DNA fragments.  This is useful information when designing PCR 
primers, as one can decide how many additional nucleotides to tack 
on to the ends outside of the restriction site.
In your specific case, the table gives the following information:
    BamHI - 97% efficiency with 1 base pair between the restriction site 
and the terminus of the fragment

     HindIII - 3 base pairs   -   90% efficiency
                  2 base pairs   -    91% efficiency
                   1 base pair    -     0% efficiency

Clearly, HindIII would not cut with 0 base pairs, as in your amplified 
fragment - so you will have to clone into the PCR vector THEN cut, or 
redesign the primers.
Hope this helps,
Rebecca

Rebecca P. Schall, Ph.D.
PanVera Corporation
565 Science Dr.
Madison, WI 53711
(608) 233-9450
rebeccas at panvera.com