6 replies to this topic

[SF_HK]

I was preparing competent cells using CaCl2 and after incubating my DH5alpha for 7 hours, I got a reading of OD600: 1.3  Is this too high to make competent cells?

[hannama]

Yes, way too high. Generally if you innoculate 50ml L-broth (or equivalent rich medium) with 250 or 500ul of overnight bacterial culture (E.coli), they will reach an O.D.600 of between 0.4-0.6 after 2 or 3 hours (checking them after 1hr 45 min is a good idea though, to make sure you don't overshoot). At this point they're in exponential phase and are ideal for rendering competent.

Hope this helps,

hannama  

[GNANA]

yeah u r far ahead from the right OD...0.5 is optimal for DH5a...Also only good handling could make good competent cells irrespective to OD...good luck..

Gnana..

Edited by GNANA, 23 September 2010 - 04:29 AM.

[jelmore]

Check out this website for making competent cells. The protocol is excellent. I routinely prepare competent Top10 cells with 1x10^8-9 CFU/ug using it. Even my first time when I butchered it I got around 1x10^7.

http://openwetware.o...competent_cells

The protocol works just fine with DH5alpha and BL21(DE3) derivatives as well, as well as many others.

[bob1]

Or read the original paper from Hanahan...

Attached Files

•  Hanahan 1983.pdf   1.48MB   122 downloads

[phage434]

The original Hanahan paper you provide does not describe the protocol on Openwetware.  Having tried them all, over a period of many months, the CCMB80 protocol is the one you want to use.  The patent literature is where the real clues are provided, as linked on that page.

[bob1]

I've used them both and they seem to work equally well in my hands.  CCMB80 is essentially the same as FSB from Hanahan.