3 replies to this topic

[donny]

I am trying to make DH1 chemical competent cells using the transformation buffer found here. However, my cells look very flaky and clumpy when I try to resuspend them in the transformation buffer. I'm not sure if it's the buffer, the nature of DH1 or something that I've done wrongly. I streaked DH1 cells on LB-agar with 15ug/mL nalidixic acid, then inoculated a colony into 10mL LB with 15ug/mL nalidixic acid for overnight growth at 37C. 1 mL of the overnight cells was then added to 400mL of LB and grown to OD600 0.4. The cells were contrifuged at 4000g for 3min in 50mL falcon tubes. This was when I added the transformation buffer to the pellet and had difficulty resuspending the cells. I tried TSS buffer and got the same results.

Has anyone have such experience too?

EDIT: Just to add that I just took the clumpy part and diluted with water. They seem to resuspend very well. What's going on?!?!

Edited by donny, 02 August 2012 - 06:21 PM.

[bob1]

I use this protocol routinely (last used yesterday) and it works fine for me.  The cells can be fairly hard to resuspend, but that is pretty typical for E. coli.

I'm not entirely sure why you are using an antibiotic for your cells, but that may have something to do with your problem.

[donny]

Thanks Bob! I'm using antibiotic to minimise contamination by other bacteria since DH1 is resistant to nalidixic acid (though growth is much slower). Is that unadvisable? I have done so with XL10 gold to make electrocompetent cells and it was ok. This is actually my first time making chemical cells as I've always used electrocompetent cells.

[bob1]

I've never had problems with contamination of my stocks with other bacteria, using standard microbilogical techniques (flaming etc.), so I don't see that you would have similar problems unless you are using heaps of different strains all at the same time, evne then proper technique should reduce the risks to a minimum.

With your seed stocks, you will be putting so many bacteria into the culture that they should be pretty well all your desired strain even if you did manage to contaminate it somehow.