Genomic ß-Lactamase? Blue White screening not working - (Jul/03/2014 )

Hi everyone,

I'm having some trouble regarding my cloning procedure.

In the past we used to have some XL-Blue competent E.Coli for transformation. Now we switched to another commercial strain

(http://tools.lifetechnologies.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf)

For cloning I always subclone my PCR products first into pGEM-T vector, transform my bacteria and use blue-white screening to select positively transformed clones. Therefore I have LB-AMP plates containing X-gal/IPTG. With the old XLblue bacteria the transformation always went well, white clones contained the insert and everything was fine.

With the new DH5a strain after pGEM-T transformation I got lots of white colonies (200 white 5 blue) but when I screen the white clones - they have no insert. I tried it now several times, I checked the plates, made new ones, used new Xgal/IPTG but no change. So could anyone please tell me what wents wrong here. I'm not able to proceed with my cloning for 3 weeks now and I have absolutely no clue why the PGEM-T system does not work here anymore. It must definitely have to do with the new E. coli I have to use now...but what's the secret behind it?

Subcloning Efficiency™ DH5α™ Mehr anzeigenCompetent Cells contain the following genetic markers resulting in these benefits:

• lacZΔM15 for blue/white color screening of colonies on plates containing X-gal or Bluo-gal
• recA1 ensures increased insert stability and prevents unwanted recombination
• endA1 improves the yield and quality of plasmid DNA prepared from minipreps
• DH5α™ competent cells support replication of M13mp vectors but do not support plaque formation
 

I would be trying a control strain which you knew was making the lacZ fragment.

X-Gal is inferior to S-gal as a selection marker. You might find S-gal generally works better for this selection (probably not your problem).