Trivial transformation improvement
hzhen at freeuk.com
Wed Feb 23 20:55:53 EST 2000
Rafael Maldonado wrote:
> Not everything in Science is thinking. There is also knowledge.
> Bacteria don't grow well at high cell concentration, as in the recovery
> step after trasformation.
Pardon my language, but this is bollocks! The cells can grow well at high cell
concentration, everything depends on nutrients and growth conditions. IIRC, it is
in fact the exact opposite, cells don't grow well at very low cell concentration.
If you start a cell culture with very low amount of cells, the lag phase will be
much longer than would be expected with the cell doubling time longer than at log
phase. Coli likes the company of other coli, OK? If you grow coli in a good
fermenter, you can get OD of ~100. If you look into your plates, what do you think
you are seeing in the colonies? Discrete individual cells well separated from each
other? No, it's coli squashing their bums against each other, piling high thousands
deep, fimbrae all akimbo.
Note that the genius who said it originally used incubation of 1-2 hours, I would be
very much surprised if there isn't at least a doubling of cells.
> Doubling times in a growing culture cannot be
> extrapolated to cells recovering from a transformation step,
> concentrated 10 to 50 fold of a overnight culture,
Oh well, let's see. Standard transformation protocol, cell harvested at OD ~0.6,
cell finally concentrated to ~1/10-1/25 (protocols varies, I used 1/12) of original
cell culture volume in transformation buffer, therefore let's say prepared competent
cells at density of OD of 10. Standard transformation procedure: 50 ul of cell
transformed, add SOB to 1 ml, therefore final cell OD ~ 0.5. What's that you are
saying about 10 -50 fold concentration of overnight culture?
> and after a sort of
> stress condition.
> So, one hour recovery incubation does not give you siblings after transformation.
Excuse me while I put my appreciation of your superior knowledge on hold for the
time being. Until you produce some references that is. Here's a reference for you
in the mean time with a quote from the abstract:
Huff et al, (90) Biotechniques 9(5), 570-2, 574, 576-7
... and an unchanged or even increased transformation efficiency when the
preplating incubation step was omitted.
> Rafael Maldonado
> Divison of Genetics
> University of Alicante (Spain)
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