Cloning PCR products

Warren Wakarchuk num208ww at MBDS.NRC.CA
Wed Jan 29 21:46:21 EST 1992


In article <9201280146.AA11363 at molly.sdsc.edu>, mws%molly at SDSC.EDU (Michael W. Smith) writes:
>I am designing a new set of primers for PCR analysis and planning to use
>restriction enzyme sites for very rare cutting enzymes.  The products would
>then be forced into a vector at those two sites.  
>
>Does anyone have tales of great success or woe with the ClaI or SacII??
>
>I am especially interested in knowing the number of extra bases
>incorporated into the 5' end of the PCR primer.  
>
>Thanks for any help
>mike
   Mike, I also have not tried ClaI or SacII but tried NdeI and XbaI
which had 8 bp tails at the 5' end.  The NdeI would not cleave at all
until I actually cloned the fragment blunt ended then exised my Nde
Xba fragment.  The Xba cleavage worked without incident.

Warren
Dr. Warren Wakarchuk   Prefered email: wakarchu at biologysx.lan.nrc.ca
                       Alternate     : NUM208WW at MBDS.NRC.CA
IBS-Protein Structure and Design
National Research Council Canada
Ottawa, Ont.,
Canada K1A 0R6   ***THESE ARE MY OPINIONS NOT THOSE OF NRC***



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