screening lb agar plate for bacteria expressing a protein

Wolfgang Schechinger Wolfgang.Schechinger at med.uni-tuebingen.de
Fri Mar 17 13:37:18 EST 2000


Hi all, 
currently I try to get a cDNA into an expression vector. (a 
modified pMAL-c2x from NEB). The cDNA is coding for His6 and a myc 
epitopes and for GFP. For experts: malE is not functional anymore.

Since I got too many colonies to perform minipreps and RE digests on 
every single one, I thought I would plate a replica on IPTG-plates 
and check for protein expression first. Then, I would transfer the 
bacteria as replica on a nitrocellulose membrane and make an ECL
on the blot with e.g. anti-myc.

Does a protocol exist for that procedure? The main problem probably 
is how tho crack the bacteria and glue their proteins to the 
membrane. Is there something easier (like flashing with blue light on 
the plate and see the expressing bugs glow green)?

All input is welcome!

Cheers,

Wolfgang
-----
This message is encrypted. Use your brain to decode it.
-----
Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
University of Tuebingen, Germany
email: wolfgang.schechinger at med.uni-tuebingen.de 
wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
-----
usual disclaimers apply 
-----
---




More information about the Methods mailing list