Cutting PCR-created restriction sites

futers at biovax.leeds.ac.uk futers at biovax.leeds.ac.uk
Mon Mar 4 12:39:45 EST 1996


In article <watson_j-2802961628150001 at s107-unknown223.wf.bms.com>, watson_j at bms.com (A. John Watson) writes:
>> Marc J. Mass, Ph.D. 
>> (mass at am.herl.epa.gov) 
>> wrote: : We have used PCR to create a BclI restriction site not
>> : normally present. The fragment is about 500 bp long, and
>> : after digestion it should be about 420 bp. We have found 
>> : that the PCR product is not cutting to completion with Bcl (about 90% of it
>> : is cutting).  According to our calculations we are certainly
>> : using enough enzyme.  Does anyone have experience with
>> : getting PCR-created restriction sites to cut.
>> 
>> 
>> : MJ Mass, Ph.D.
>
>Well, I'd be satisifed with 90%  ;^)   Is there some reason you need to
>digest to completion?  BTW, if you check the back of the New England
>Biolabs catalog you'll find a list of oligo cleavage results for a number
>of different enzymes -- some work well, some don't.  In general, it's all
>in how much flanking DNA the endonuclease likes to have surroiunding the
>recognition site.
>
>Cheers,
>
>AJW
>
>----------------
>John Watson
>Bristol-Myers Squibb Co.
>watson_j at bms.com
>---------------------------------------------------------------------
>"If you're not part of the solution, you're part of the precipitate."
>---------------------------------------------------------------------

I am also looking at a Bcl I polymorphism and also only get about 90%
digestion.  I just ignore the faint upper band in the homozygote.
                  Simon.




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