This is a cached page for the URL (http://www-db.embl-heidelberg.de/jss/servlet/de.embl.bk.wwwTools.GroupLeftEMBL/ExternalInfo/stewart/ETprotocols.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Group Left EMBL Heidelberg

The Stewart Group left EMBL Heidelberg

The information on this page is not maintained at the EMBL any longer.
Address Info (as of 07/2001): Technical University Dresden, c/o Max Planck Institute for Cell Biology and Genetics, Pfotenhauerstrasse 110, D-01307 Dresden, r2s12the hompeage address (as of May 2002) is www.biologie.tu-dresden.de/stewart/index.htm



For your information we provide the old content below. Regards, webmaster@embl-heidelberg.de.

ET cloning protocols

Protocols for ET recombination.

    Oligo design

    PCR reaction and recipient plasmid DNA preparation

    Preparation of competent bacterial cells

    Transformation

    Using Cre or Flp transient plasmids

    705-Cre and 705-Flp plasmids are based on the pSC101 temperature sensitive origin. This origin maintains a low copy number and replicates at 30oC. These plasmids will be lost from cells when they are incubated at temperatures above 37oC. In addition, Cre and Flp are expressed from the lambdaPR promoter, which expresses weakly at 30oC and strongly at 37oC. Thus 705-Cre and 705-Flp can be used to give a transient burst of expression after which they will be eliminated so the recombined product can be isolated uncontaminated by their presence.

    Using Cre or Flp expressing E.coli

    294-Cre and 294-Flp were generated by integrating 705-Cre and 705-Flp into the lacZ locus of E.coli strain, MM294 (Buchholz et al, Nucleic Acids Res. 24, 3118-9 (1996).

Author: Francis Stewart
Back to the wombat page