*** For details on how to be removed from this list visit the ***
*** CCP4 home page http://www.ccp4.ac.uk ***
Dear Raji,
I have gotten similar stuff to work also, but the right tool to this is out
there from Novagen. They sell plasmids that would allow for you to
co-transform and have separate replicons, like pCDF and pACYC along with
pCOLA or pRSF to use with the pET vectors.
But if you want to stick with your two plasmids, then I would transform with
1/2 of the "usual volume" of DNA from each plasmid and plate both on AMP
only and KAN only plates as well as the double antibiotic AMP+KAN plates. I
always use the usual dose of each the antbiotics even when I use 4
antibiotics for the strain (for 4 different plasmids). If you get each of
the plasmids to work alone on each of the control single antibiotic plates
and the co-transformed ones on the double antibiotic plates do not work,
then I would presume that something about the two plasmids are not
compatible. Then you can try and see if you can transform one plasmid and
then make that strain competent and then transform the second plasmid. Hope
this helps
Peter
----- Original Message -----
From: "Raji Edayathumangalam" <[EMAIL PROTECTED]>
To: "'CCP4 Bulletin Board'" <[email protected]>
Sent: Friday, July 07, 2006 4:45 PM
Subject: [ccp4bb]: Co-transforming two different plasmids
*** For details on how to be removed from this list visit the ***
*** CCP4 home page http://www.ccp4.ac.uk ***
Hi Y'all,
Non-xtallo query.
I have the foll. plasmids:
1) a pET21a vector (AmpR) expressing a single protein
2) a pET28 vector (KanR) containing a polycistronic construct that
co-expresses four proteins
While we contemplate sticking the fifth protein and making a single
multi-expression pET28 vector, I tried to co-transform the two plasmids by
bumping up the two antibiotics and that did not work.
If you have had success with co-transforming two plasmids that share the
same replication origin (for e.g., two different pET vectors) into E.coli
expression strains, could you pl. share your tricks and/or suggestions. Do
you have favourite strains/vectors when it works?
Many thanks.
Raji
--
Raji Edayathumangalam
Postdoctoral Associate
The Rockefeller University
Box 224. 1230 York Avenue
New York, NY 10021
