help: primer restriction sites
REBECCAS at PANVERA.COM
Wed Aug 30 10:24:51 EST 1995
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
> The enzymes are: Bam HI and Hind III.
> Frank Chen
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 P. Schall, Ph.D.
565 Science Dr.
Madison, WI 53711
rebeccas at panvera.com
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