Cutting PCR-created restriction sites

sam simons simonssp at pfizer.com
Mon Feb 19 10:55:35 EST 1996


I routinely subclone PCR products into a TA vector before restriction to 
avoid the cleavage hassle altogether. You should also check the chart in 
the back of the New England Biolabs catalogue which gives minimum sizes 
of "GC clamps" for cutting ends. 90% cleavage should be plenty to do a 
subcloning anyway. Is there some reason you need 100%?

Sam 

"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.
>
>
>
>





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