Hanahan competent E. coli (capacity vs. eff.)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Apr 20 10:18:01 EST 1995


David Logan writes:

> I am planning to prepare competent cells by the Hanahan method...
> Sambrook et al. states that the number of cells in the culture 
> should not exceed 1x10^8/ml and one should determine the relationship 
> between OD and viable cell number/ml for the strain to be used.
> ... [wants to know if this is necessary]

If you go over some critical OD, you lose eff. because the cells 
become refractory to becoming competent.  If you are way under the
critical OD (but still well into log phase), you simply have fewer
cells. The efficiency is the same, but you have less capacity; ie. you
still get good efficiency with 0.1 ng, but the point at which adding
more  DNA fails to produce more colonies comes sooner.  With Hanahan
cells this point can be anywhere between 10-100ng/200 ul cells, and it 
tends to get worse when you store them.  So if capacity is an issue in
your experiment, or if you plan to store them, I recommend just
growing up 10 x the volume of cells and concentrating them 10x as much
into TFB thus pumping up the capacity.  If I wasn't confident of where
the critical OD was, I'd take them low on purpose  (0.2-0.3 OD/ml 550)
and use the same trick to compensate.  Why play chicken when at most
you're going to squeeze out a factor of 2-3 in capacity?

The critical OD presumably reflects the multiple changes that ensue
when the cells start to run out of some nutrient.  As such it will
depend on many factors besides the strain, ie the media, and even
the extent of aeration.  So this 1x10^8/ml is simply a guideline
for where typical strains would be in typical growth conditions
at mid to late log phase.  So if all else fails, monitor the growth
curve and make sure you take them while they are still log-linear.

Hope this helps
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu  



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