1 reply to this topic

[PhD]

Hello everyone,

I have been using a 50 mg/ml stock of chloramphenicol for transformationa nd expression of Bl21 de3 PlYS in LB media. Since I didnt get expression of 2 fragments I decided to try M9 minimal media. Then I came across an antibiotic info sheet that recommends decreasing Chloramphenicol concentration and decreasing my kanamycin concentration(also needed). I was wondering why I need less of this antibiotic in the minimal media and wether decreasing the concentration in LB might affect protein expression? I have made M9 agar plates with the 50mg/ml stock so if this was harsch on the E.Coli I should probably consider increasing recovery time after trafo and plating the whole thing right?

I would really appreciate some insight on this matter, it would be really cool if I could improve my protocol by tweeking some.

Have a nice day!

[fishdoc]

PhD, on Jul 27 2009, 07:19 AM, said:

Hello everyone,

I have been using a 50 mg/ml stock of chloramphenicol for transformationa nd expression of Bl21 de3 PlYS in LB media. Since I didnt get expression of 2 fragments I decided to try M9 minimal media. Then I came across an antibiotic info sheet that recommends decreasing Chloramphenicol concentration and decreasing my kanamycin concentration(also needed). I was wondering why I need less of this antibiotic in the minimal media and wether decreasing the concentration in LB might affect protein expression? I have made M9 agar plates with the 50mg/ml stock so if this was harsch on the E.Coli I should probably consider increasing recovery time after trafo and plating the whole thing right?

I would really appreciate some insight on this matter, it would be really cool if I could improve my protocol by tweeking some.

Have a nice day!

I can't speak to your situation directly, but I know that we were using a chlor cassette in some vectors in LB media, and we had to decrease the concentration to 12.5 ul chlor, because even the resistant bacteria were not growing.

It had to do with the number of copies of chlor resistance present. When it was in a high copy number plasmid, it handled the 50 ug/ml just fine, but in either a lower copy number plasmid or on a single insert in the genome, it could only handle 12.5 ul.