Hello. I am trying to make competent cells and the transformation buffer in my protocol requires MnCl2. I don't have it, but I do have MgCl2 and am wondering if it will work the same.
The protocol also asks for 10mM PIPES disodium for transformation buffer. I don't have it and have never seen it used for this purpose, thus if anyone can enlighten me on its role and whether it is critical for making competent cells that be great!
Thank you very much!
It sounds like you're trying to make Inoue transformation buffer from this article: Article Inoue, H. , Nojima, H. & Okayama, H. High efficiency transfo...
It's been a while since I read it and I don't currently have access but I do vaguely remember that there were specific reasons why those components were chosen.
I think PIPES gave a somewhat better transformation efficiency than the other buffers tested and I think MnCl2 was pretty essential for drastically increasing the transformation efficiency compared to previous methods. You could probably get away with substituting the PIPES (but not the MnCl2), but definitely read the article to verify that.
Hello Alexandra,
Yes exactly that's the buffer I am Making. I can substitute PIPES with Hepes without the loss of efficiency, but they did not test whether MgCl2 works. They only report MnCl2 data.
Dear James,
There have been a lot of developments of protocols to prepare Escherichia coli competent cells, and the buffer you want to make is one of these - but washing cells with CaCl2 can produce reasonable competent cells too (though this does date back to the late 1980's)!
Protocol:
1. Inoculate (1%) overnight culture in LB broth and grow to mid-log phase at 37°C.
2. Harvest the cells by centrifugation at 10K 5 minutes.
3. Resuspend cells in 0.1 volumes of ice-cold 50mM CaCl2 and incubate on ice for thirty minutes.
4. Harvest the cells by centrifugation at 10K 5 minutes.
5. Resuspend cells in 0.05 volumes of ice-cold 50mM CaCl2.
6. Keep on ice for (minimum) of 1 hr., preferably overnight.
7. Add glycerol to a final concentration of 15% (2.5ml 50% to 5ml cells).
8. Freeze at -70°C in aliquots. Competent cells stored in this way will remain viable for 12 months or more.
9. For transformation, thaw the cells on ice.
10. Mix 100ul cells with DNA and incubate for thirty minutes on ice.
11. Heat shock at 42°C for three minutes.
12. Add 900ul LB broth and incubate at 37°C for one hour.
13. Plate 100-200ul aliquots on selective plates.
Regards, Andrew
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