Recombinant phage antibody system

ijiwaru at wheel.dcn.davis.ca.us ijiwaru at wheel.dcn.davis.ca.us
Tue Apr 2 14:27:21 EST 1996


In article <315F6A6E.50 at mail.ncku.edu.tw>, Chao-Liang Wu
<wumolbio at mail.ncku.edu.tw> wrote:

> Dear Colleagues,
> 
> I would appreciate if anyone out-there can tell me his experience about 
> the preparation of "Coliclonal Antibody".  Are Pharmacia's products good 
> enough to a beginner to produce coliclonal antibody ?  
> 
> Thank you in advance for your assistance.
> 
> Chao-Liang Wu
> 
> Department of Biochemistry
> National Cheng Kung University Medical College
> Tainan, Taiwan

Dr. Wu,

I am currently using a modified pCANTAB5E vector from Pharmacia's RPAS. 
The problem with their "system" is they provide the vector already
digested with SfiI and NotI and will not provide it outside of the kit. 
The lac promoter is quite leaky and will yield high levels of pIII fusion
if you do not use glucose to quiet the promoter (for our purposes 0.25%
works quite nicely YMMV).  I haven't used any other parts of the kit
because they did not suit our current needs so I cannot comment on the PCR
primers or the sequencing primers that come with the kit.  Antibodies
against M13 phage are not provided and I found this to be quite a set back
since I could not initially detect the pIII protein produced by the vector
even when grossly overloading the gel and silver staining.  I wish you the
best of luck.

Lyle Najita
Plant Pathology
University of California - Davis



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