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Starter culture dilution (preparation of competent cells)


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#1 lab_member

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Posted 25 January 2010 - 01:02 AM

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

#2 KAUSHIK THAKKAR

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Posted 25 January 2010 - 02:48 AM

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


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. :D

Hope this helps.

Best regads,
Kaushik
;)

Attached Files



#3 leelee

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Posted 26 January 2010 - 11:09 PM

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!

#4 lab_member

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Posted 28 January 2010 - 12:40 AM

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


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. :lol:

Hope this helps.

Best regads,
Kaushik
:blink:


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.




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