No colonies when transforming BL21 coli w/ chloramphenicol selection - (Jun/03/2006 )

Hi all,

I am trying to transform the protease-deficient E. coli strain BL21 with pRARE (Novagen - it's a plasmid that codes for rare tRNAs to assist with heterologous protein expression, selected on chloramphenicol).

The plasmid is supercoiled, so should be trivial to transform, but it just doesn't want to go in! I used a positive control (an ampR plasmid, selected on amp) and the cells transformed OK. Also, DH5-alpha cells transformed OK with pRARE, although there were fewer colonies that I expected on the Chloramp. plates.

Is there any difference between transformation procedure with Amp and Chloramp.?

prepare plates using half the chloramphenicol concentration.
Let the bacterias recover after transformation for at least 1h (better 90')

I think Fred's got it -- how long did you let your transformants express without selection (to overcome phenotypic lag) before plating them on the chloramphenicol plates?

Chloramphenicol is a very stressful antibiotic which is why most labs resort to plasmids with amp/kan resistance for protein expression.

I would let them grow for 24 hours and reduce the concentration in half.

-Matt

Thank you to everyone who replied, I followed your recommendations and got about 200 colonies this time.

I reduced the chloramphenicol concentration to an intermediate concentration of 68 ug/ml (about 2.5 x the amount Maniatis recommends for stringent plasmids but 2.5-fold less than recommended for relaxed plasmids). I also increased post-transformation recovery time to 90 minutes.

It must have simply been a tricky combination of a stressful antibiotic with a "sick" protease-deficient strain.

Cheers,

MP