3 replies to this topic

[lab_member]

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

[KAUSHIK THAKKAR]

lab_member, on Jan 25 2010, 02:32 PM, said:

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.

Hope this helps.

Best regads,
Kaushik

Attached Files

•  Competent_cells_Prep_Protocol.pdf   38.36K   486 downloads

[leelee]

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!

[lab_member]

KAUSHIK THAKKAR, on Jan 25 2010, 06:48 PM, said:

lab_member, on Jan 25 2010, 02:32 PM, said:

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.

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.