can we prepare Copmetent cell by calicum cloride with 0.09 OD - Cp cells with 0.09 OD (Feb/15/2009 )
Hi All ,
I want to understand why we are allways preparing competent cell of Ecoli when we grow it till arrive OD into (0.5-0.6) ?
Because my experiment purpose need to very little cp cells ,So i can get a good result to my experiment when i prepare Competent cells with 0.09 optical density ,Is this OD. scientifically possible or not ?
Also due to my transformation result in broth media , i got declining in bacterial growth rate !!!! when the growth arrived to o.34 OD. but it has to continue increasing in bacteria growth like in Cp cell with 0.5 OD.
Any help please !!!!!!!!!!!
Dawood80 on Feb 15 2009, 12:53 AM said:
I want to understand why we are allways preparing competent cell of Ecoli when we grow it till arrive OD into (0.5-0.6) ?
Because my experiment purpose need to very little cp cells ,So i can get a good result to my experiment when i prepare Competent cells with 0.09 optical density ,Is this OD. scientifically possible or not ?
Also due to my transformation result in broth media , i got declining in bacterial growth rate !!!! when the growth arrived to o.34 OD. but it has to continue increasing in bacteria growth like in Cp cell with 0.5 OD.
Any help please !!!!!!!!!!!
i don't think it is feasible, cell density at 0.09 OD is very low, usually a portion of cell will die during transformation (especically in electroporation, the cell density must be high as a large amount of cell will die after electrical shock) and that will give you extremely low recovery of the cells. I don't get the last point you mentioned.
Whether you can even accurately measure an optical density of 0.09 depends on the specifications of your spectrophotometer, in particular its photometric accuracy and its linear dynamic range (defined as the concentration range over which absorbance and concentration remain directly proportional to each other).
The purpose of letting bacteria grow till o.5-0.6 OD is to catch them in the log phase. In early log phase, the cells are healthy and 'raring to go'. They are metabolically active and people (in the early era of recombinant DNA technology) apparently found that cells in this phase are much more receptive to being made competent and teking in DNA than in any other phase. Of course, this is an empirical observation.
So, the purpose of growing till Od 0.6 or so is not for the quantity of cells but rather for their quality.