IUBio

Pharmacia phage display kits

Deb Britt Deborah_Britt at brown.edu
Mon Sep 25 15:01:01 EST 1995


In article <kkMRAbu00iUvI8kFp2 at andrew.cmu.edu>, "Morley O. Stone"
<mscj+ at andrew.cmu.edu> wrote:

> I'm interested in anybody's experience using the recombinant phage
> antibody system sold by Pharmacia. Did it work as advertised? How easy
> was it to purify mRNA from the hybridoma cells? How well did their
> primers work? Did one module work better that the other? etc. 
> Any helpful information you could give me regarding this kit would be
> greatly appreciated.
> Thanks in advance,
> Morley

I've been working with the Pharmacia kit for about three months, but
haven't yet successfully gotten a functional ScFv.  In my experience
*nothing* works as well as advertised.  I found with this kit that mRNA
prep was easy, and the primers work well.  The assembly PCR reaction is
tricky.  You may need to purify your heavy and light chain bands more than
they suggest (i.e., use etOH precip or a gene-clean type of step, instead
of their quick spin column), and set up a couple of reactions to get the
optimum result.  I had a hard time with quantity - getting enough PCR
product to clone.  Cloning and phage expression are straightforward, but we
seem to have a problem with the panning.  We get colonies to pick, but none
seems to have what we want. We are using cells or cell extracts for
panning.  You may have more luck if your antigen is highly purified.  I am
disappointed that it is taking so long, but I think it is just a matter of
working through the technical problems.  I must say Pharmacia technical
support has been very helpful when I have called them.

Good luck!

Deb

Deborah Britt, Ph.D.
Medical Oncology
Rhode Island Hospital/Brown University 



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