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Amount of inoculum?


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#1 ram

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Posted 03 November 2009 - 06:41 AM

General practice for recombinant protein expression in E. coli is
1. Transform the cells with the recombinant plasmid and spread on LB agar plate
2. Pick up a colony in eg. 5 ml LB. Grow for few hrs
3. Use part of this culture eg. 0.5 ml for culture initiation in 100 ml LB.
4. And then go ahead till the desired OD achieved.
My doubt is for the step 2 and 3. Why are the cells initially grown in smaller amount of media? And then only part of the grown culture transferred to larger volume of media? Why not to grow the colony directly in 100 ml LB? Or why not to use whole culture obtained after step 2 for step 3?
Has anyone ever got different/negative results after doing so?

Edited by ram, 03 November 2009 - 06:42 AM.

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#2 Prep!

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Posted 03 November 2009 - 09:13 PM

hi ram.. i m neither a microbiologist nor do i have any experience in fermentation... bue still i think the spreading is done to get isolated colonies.. and one colony is picked up so that you are sure you have the single species of wanted bacteria.
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#3 swanny

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Posted 04 November 2009 - 04:39 PM

Typically, the first culture in 5 ml is done overnight, rather than "a few hours". By the next day, the cells will have gone from the growth phase, where they divide every 20-30 minutes, depending on the strain, temperature and other factors, to the vegetative phase, where they stop actively dividing rapidly.
You only add a small amount of this culture to the large volume of media because you don't want the cells to reach that vegetative phase too quickly, and because the 5 ml culture will have too many metabolic by-products that inhibit protein expression. 0.5 ml in 100 ml is a 1/200 dilution, which leaves a lot of growth potential.
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#4 ram

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Posted 05 November 2009 - 03:11 AM

Typically, the first culture in 5 ml is done overnight, rather than "a few hours". By the next day, the cells will have gone from the growth phase, where they divide every 20-30 minutes, depending on the strain, temperature and other factors, to the vegetative phase, where they stop actively dividing rapidly.
You only add a small amount of this culture to the large volume of media because you don't want the cells to reach that vegetative phase too quickly, and because the 5 ml culture will have too many metabolic by-products that inhibit protein expression. 0.5 ml in 100 ml is a 1/200 dilution, which leaves a lot of growth potential.


Thats perfect...
Thanks swanny :o
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.




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