Competent Cells Using CaCl2

[gandalfthegrey]

Hello everyone,
I have another problem I am trying to make competent cells using Calcium chloride CaCl2 however I am not getting good competent cells.

Does anyone know any tips or can post a good cacl2 protocol?

thank you all in advance

gandalf

[aftabac]

Hi
i do prepare competent cells with following procedure with good results and i hope it will also help you.
-inoculate 5 ml of LB broth with single colony of any bacterial strain you want to make competent (E.coli), and incubate it on shaker at 37oC for overnight.
-on next day inoculate 10-15 or (as your requirment) of LB broth and incubate again on shaker at 37o C untill O.D. 550nm reach 0.2-0.3 (it usually take 2-3 hrs)
-place the culture on ice for 5 min and then transfer to pre-cooled falkon tubes.
-centrifuge the culture at 6000 rpm for 5 min at 4 oC.
-Discard the supernatent
-Gentally resuspend the pellet (cells) in ice cold 50mM CaCl2 (for 15 ml starter culture, add about 20 ml of CaCl2) and leave on ice for 20-40 min (40 min is preferred)
-Centrifuge the cells again at 6000 rpm for 5 min at 4 o C.
-Discard the supernatent and gentally resuspend the pellet in ice cold 50mM CaCl2 (1-2 ml) and store on ice untill use.
it is better to use immediatly for better results.

best regards
aftab ahmad

[miss-pcr]

try to check the concetration of CaCl2