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Competent cells- just won't work!

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Postby canalon » Mon Sep 13, 2010 5:59 pm

Don't know about the scaling down, never tried it. But considering the cost of reagents vs the time taken by repeated failure, maybe worth trying preparing 40 tubes... or even 20.

However I am surprised that you have problem resuspending the cells. My experience is that when washed in a lot of water (and I do not know why) it is quite hard to get a pellet that is solid. they tend to resuspend a bit too quickly.

A pasteur pipette will be a bit gentler (larger opening), or flame it to form a glass sphere at the end and gently use that to mix your suspension.

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Re: Competent cells- just won't work!

Postby kimsmarkin » Wed Sep 22, 2010 7:10 am

Electroporation has worked for the same stem. Regarding the chemical transformation, I am not sure when I recently had to move from one water bath to a heating block, maybe I did not have the right temperature.
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Re: Competent cells- just won't work!

Postby pavanibeesetty » Tue Dec 14, 2010 12:05 am

I have been using Zymokit to make competent cells and I have the same problem as u mentioned. I have been trying to make competent cells of a uropathogenic strain of E.coli and the cells I get are not that competent. But i was able to successfully transform them with pUC19 but am not able to transform with my Kan R plasmid. The cells do not have any other plasmid. I think maintaining 4C is very imp during making this competent cells as similar trial which i performed before didnot work (which i think because at some point the temperature of the competent cells is increased to above 4C while prep). But the second time where i took great care of temp I was able to transform atleast my positve control (pUC19).
And may be mixing the cells gently might also have some effect on the cells.
One more suggestion given to me by the tech support guy of Zymocompany is to grow the cells in Zymobroth with a very low initial OD. (I donno if any one is using this kit. But i just wanna help the people who has faced my situation.
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