no colonies on my ampi/LB agar plate
 

I'm trying to do cloning a specific gene, but after I optemized my bacterial CFU to get a colonies on LB agar plates without antibiotic, I did transformed my competent cells DH10B by heat shock with positive control PUC 10pg/ul

10ul control with 100ul competent cells incubated on ice for 30 min then in water bath 42C for 90 sec then on ice again for 2 min then I add SOC medium 400ul and incubated on 37C / 1 hours, I then cultured the bacteria on LB agar plat with antibiotic and added x-gal, IPTG after 24 hours in 37C incubation:

no colonies on the plat

Note: when I did transformed high conc of the bacteria I get a bacterial lawns on the plate without any color or colonies

Re: no colonies on my ampi/LB agar plate
 

I don't know about DH10B cells, but for DH5alpha cells, it is very important to only heat shock them for 30 seconds. Secondly, make sure the concentration of your antibiotic plates is correct. If you are using Ampicillin, it should be roughly 70ug/ml. Hope that helps. Good luck.

Re: no colonies on my ampi/LB agar plate
 

thank you DrIHC, my ampicilling conc is 50ug/ml. I will try the 30s heat shock, do know about electroporation??

Re: no colonies on my ampi/LB agar plate
 

thank you finally I got a transformed E.coli when I shocked it just for 35 s but I did determined first the CFU of the bacteria first,,, although i never read about it as a cloning protocol..I guess the to much CFU was on of the inhibition factor,,,,

did any one try to dilute the bacterial stock before the heat shock????