PCR Restriction Site engineering

choie01 at mcclb0.med.nyu.edu choie01 at mcclb0.med.nyu.edu
Sun Jan 23 22:50:56 EST 1994


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



More information about the Methods mailing list