Transformation by CaCl2 method - (Mar/08/2006 )
I am interested to know during transformation procedure, after we prepare the competent cells by CaCl2 method , we give heat shock treatment at 42 degree C for 90 seconds and then after cooling the tubes we add 800 ul of LB medium without antibiotic and then incubate the tubes for 1 hour at 37 degree C. After that we centrifuge and get the cells pellet and this time should we add 200 ul of LB medium without antibiotic or with antibiotic. I wanted to know if we are doing plating on antibiotic containing plates then do we need to add the antibiotic in the LB medium before we plate them out?
You don't need to add antibiotics.
the reason you centrifuge is only to concentrate the bacteria.
then you will select your bacteria on the antibiotic containing agar. (the 200 uL wont't dilute so much the antibiotics)
Actually I centrifuge, discard some but not all of the medium and resuspend the bacteria in the medium left, so my bacteria are concentrated, and then I plate them on ATB agar.