competent cells - OD600 reading (Sep/23/2010 )

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?

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

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..

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.org/wiki/TOP10_chemically_competent_cells

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

Or read the original paper from Hanahan...

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.

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