Starter culture dilution (preparation of competent cells) - I don't know how much should I dilute my starter culture... (Jan/25/2010 )
I am planning to prepare some electrocompetent e.coli for my lab. The protocol in my lab dilutes the starter culture into large culture by about 1:20, but another protocol found in openwetware (for chemically competent cells though) suggest to at least dilute by 1:100.
I understand that the purpose of dilution is to bring the e.coli to lag phase so they can re-enter log phase again during culturing where they are harvested. I am not sure whether a 1:20 dilution is enough to bring the e.coli back to log phase as I have no idea the OD600 of e.coli in lag phase (strain HB101, in SOB media).
So should I stick to the 1:20 dilution? Or should I do a 1:100 dilution?
Thank you
lab_member on Jan 25 2010, 02:32 PM said:
I understand that the purpose of dilution is to bring the e.coli to lag phase so they can re-enter log phase again during culturing where they are harvested. I am not sure whether a 1:20 dilution is enough to bring the e.coli back to log phase as I have no idea the OD600 of e.coli in lag phase (strain HB101, in SOB media).
So should I stick to the 1:20 dilution? Or should I do a 1:100 dilution?
Thank you
Hello,
We generally put 2 ml of starter culture to 100 ml of LB. (close to 1:50)
Refer the attached file.
I should say it is not hard & fast. I think if you add 1:100, only difference it will make that it wud take more time for it come till OD 0.45, and if dilute it less 1:20 it will take less time.
I would advice to stick to 1:50 or 1:100, as there would be less stationary phase cells in them.
Hope this helps.
Best regads,
Kaushik
Why not just do one of each?
And then use the one which reaches OD600 in no more than 2-3 hours.
Always works for me!
KAUSHIK THAKKAR on Jan 25 2010, 06:48 PM said:
lab_member on Jan 25 2010, 02:32 PM said:
I understand that the purpose of dilution is to bring the e.coli to lag phase so they can re-enter log phase again during culturing where they are harvested. I am not sure whether a 1:20 dilution is enough to bring the e.coli back to log phase as I have no idea the OD600 of e.coli in lag phase (strain HB101, in SOB media).
So should I stick to the 1:20 dilution? Or should I do a 1:100 dilution?
Thank you
Hello,
We generally put 2 ml of starter culture to 100 ml of LB. (close to 1:50)
Refer the attached file.
I should say it is not hard & fast. I think if you add 1:100, only difference it will make that it wud take more time for it come till OD 0.45, and if dilute it less 1:20 it will take less time.
I would advice to stick to 1:50 or 1:100, as there would be less stationary phase cells in them.
Hope this helps.
Best regads,
Kaushik
Thanks a lot for the protocol! I diluted by bacteria 1:100 and actually it didn't take that long to grow up to the desired OD600, only 1.5 hrs, so I think I just stick to 1:100.