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BL 21 cells


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#1 scary

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Posted 24 July 2009 - 09:28 PM

Hi,

I have a problem in understanding if I can make glycerol stocks of transformed BL21 cells for future expression.

I have my NPT II gene in pET15b and I have transformed my clone into BL21(pLys) cells.My question is can I make glycerol stocks of these cells and express them some time later??

Thank you in advance.

-Scary

#2 klinmed

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Posted 25 July 2009 - 02:30 AM

Hi,

I have a problem in understanding if I can make glycerol stocks of transformed BL21 cells for future expression.

I have my NPT II gene in pET15b and I have transformed my clone into BL21(pLys) cells.My question is can I make glycerol stocks of these cells and express them some time later??

Thank you in advance.

-Scary

The answer is yes and no.

In expression host strains (containing the DE3 lysogen) pET plasmids are quite "leaky". Thus, if your expressed protein is even slightly toxic you risk selecting for plasmid with deletions etc. The risk can be limited by minimizing the total time the bugs are grown.

Ideally, you should freshly transform your plasmid into BL21(DE3) pLysS before each expression. Just set up the starter culture with the whole transformation mix (ie no need to plate on agar).

Alternatively, if your product is not too toxic, you can make 1 ml glycerol stocks from a (freshly transformed) mid log culture. Use one 1 ml stock tube to replace the starter culture for each expression.

If your protein in not toxic you MAY be able to inoculate a starter from a glycerol stock as you would normally do for plasmid preps etc. But you need to test if this does not lower expression levels.

Hope this helps.

As an after thought have included the attachment. This is the "gold source" for info on the pET system.

Attached Files


Edited by klinmed, 25 July 2009 - 03:10 AM.


#3 ram

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Posted 25 July 2009 - 05:38 AM

I am just starting my expression experiments. I cloned my gene in expression vector pASK-IBA7 and transformed in E. coli (Top10, BL21 and DH5alpha), picked up the colonies from transformation plate for culturing and made glycerol stocked of it. Now, since it is recommended to use the single colony from maximum 1 week old plate, whenever I wish to do expression experiment, I directly spread the glycerol stock (0.5 ul) onto the agar plate and use colony grown therein for initiating the culture of expression.
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#4 klinmed

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Posted 25 July 2009 - 07:13 AM

I am just starting my expression experiments. I cloned my gene in expression vector pASK-IBA7 and transformed in E. coli (Top10, BL21 and DH5alpha), picked up the colonies from transformation plate for culturing and made glycerol stocked of it. Now, since it is recommended to use the single colony from maximum 1 week old plate, whenever I wish to do expression experiment, I directly spread the glycerol stock (0.5 ul) onto the agar plate and use colony grown therein for initiating the culture of expression.

That may be fine with pASK-IBA7 which has a fairly "weak" tet opertator/promotor but is not a very good one for the pET series of vectors which use strong T7 promotors.

#5 scary

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Posted 28 July 2009 - 08:13 AM

I am just starting my expression experiments. I cloned my gene in expression vector pASK-IBA7 and transformed in E. coli (Top10, BL21 and DH5alpha), picked up the colonies from transformation plate for culturing and made glycerol stocked of it. Now, since it is recommended to use the single colony from maximum 1 week old plate, whenever I wish to do expression experiment, I directly spread the glycerol stock (0.5 ul) onto the agar plate and use colony grown therein for initiating the culture of expression.

That may be fine with pASK-IBA7 which has a fairly "weak" tet opertator/promotor but is not a very good one for the pET series of vectors which use strong T7 promotors.



Thank you Klinmed for the suggestions and also for the manual.

-bye




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