>I meant cold-sensitive by cs. I have not caculated how many divisions
XL1-blue and DH5alpha are going through, but I am pretty sure they are
not dividing efficiently. Every other strain that I have tried to grow
at 18C (SURE, MC1066, C600) I need to do at least 1/20000 dilution at 5pm
to be harvesting at similar density as XL1-blue and DH5alpha. I
got 10^8 for MC1066 and C600 and 10^7 for SURE, but not 10^9. I was
wondering if high effeciency that I get sometimes with XL1-blue and
DH5alpha had anyting to do with inability of these two strains to grow at
Aha, this adds something to my list. By the way, in your hands exactly the
same strains as those known to be efficiently electroporated are relatively
inefficient in the Inoue protocol. All this discussion made me re-read
the Inoue paper carefully and there are some things in it I do not understand.
I have been using a modification of the Hanahan protocol with
XL-1 since 1984 always getting around 10E7 -10E8. And we have been very
careful in selecting reagents and always used 18 Meg water. The trouble
only started when I got some fragments to clone which were clearly unstable
in XL-1, so I had to try a variety of other strains only to find that certain
strains are just plain reluctant for transformation, even if you
try to "optimise" the protocol, changing incubation time in TFB, heat-shock,
plus-minus DTT etc. The Inoue paper openly contradicts Hanahan's in such
issues, as the use of glycerol, omission of rubidium salts, and independence
of the heat shock on temperature in a very broad range. And, they advocate
the use of plastic tubes for transformation experiment. On the other hand,
if we recall Hanahan's paper, he makes a big point of the water quality (
which I second, as there are places on this planet, where a Milli-RO machine
shuts down every spring because it is clogged by polluted water coming with
the melting snow ( or in autumn, following the harvest and defoliation season
), while other places may do well with a simple distiller.
There is no mention of the water quality in Inoue et al. Is it by default
very good (everybody has a MilliQ machine, right?) or it does not really
Then, the heat shock is apparently different in plastic tubes and in glass
tubes ( Hanahan also makes a big point of this ) and IT IS STRAIN DEPENDENT.
And finally, if you really look at Fig.3 in Inoue paper, there is no real
dependence of competence on the cell density at 18 C (<2-fold)!
So, a new proposed summary is:
1. If you have one strain to work with, grow it a RT, use whatever
transformation protocol you like (Hanahan, Inoue, Mike Scott, Cheung etc) and
you will have to work out small details yourself anyway ( water quality,
reagent sources, proximity of the centrifuge and the freezer to your
bench etc )
2. When in doubt, electroporate! But you will have to desalt your DNA first,
e.g. by precipitating with 10 vol n-butanol. And it is expensive, if done
3. If you are rich, buy (them) from Stratagene.
Have a nice day, everybody.
-------------- next part --------------
Alexander Kraev, PhD Internet: kraev at bc.biol.ethz.ch
> Lab.of Biochemistry III Phone: 0041-1-632-31-47
> Swiss Federal Inst. Of Technology FAX: 0041-1-632-12-13
> Universitaetsstr. 16 URL: http://188.8.131.52/kraev.html/> CH-8092 Zurich