PCR Restriction Site engineering

Hinayana Bawagan bawagan at umdnj.edu
Mon Jan 24 07:45:15 EST 1994


choie01 at mcclb0.med.nyu.edu writes:

>Greetings Netters,

>I was wondering if there was a conventional wisdom that people follow
>when engineering restriction sites onto the ends of PCR products by
>appropriate primer design.  That is, does anyone have a favorite
>restriction site(or pair of restriction sites) that they regularly
>design into their PCR primers for directional cloning?  Have you had
>any problems with end-cutting with your restriction digests?  How much
>do you purify your PCR product(from Taq, primers, buffer, etc) before
>you digest?

>I have asked around our department and have gotten as many replies to
>this as people that I've asked.  Also, I have consulted Erlich's PCR
>Technology:  Principles & applications, which was helpful but did not
>offer any specific guidelines for this type of genetic engineering. 
>Is this process entirely empirical?

>Any and all suggestions are welcomed and would be greatly appreciated,
>either on the Net or by e-mail.  Thanks ahead of time for your
>consideration of this request.

>Elmer Choi
>New York University Medical Center
>Department of Pathology
>e-mail:  choie01 at mcclb0.med.nyu.edu

     Restriction sites are preferably unique, not found in both insert
and vector unless one can make partial digestion work.
     There is a section in the New England Biolab (NEB) catalogue  on the
efficiency of restriction cutting of certain enzymes when the recognition
sites are at the ends of linear DNA.  One has to add extra nucleotides
usually GC's like for example, Bam HI.  Also I would put what is called
a clamp at the ends of the PCR product. I have used AAA as a clamp.
     I usually do a PCR reaction of 100 ul and I used 10 - 15 ul to analyze
in an agarose gel if I got the piece, I usually used 50-75 ul for digestion,
then gel purify it and clone into an appropriate vector for amplification,
sequencing and expression.  After gel purification, I would like to be able
to see the piece if I run a small aliquot for estimation of concentration 
for ligation. 



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