Frank O. Fackelmayer
Frank at Fackelmayer.de
Mon Aug 13 03:20:35 EST 2001
> I have a question about transformation. The lab I'm working in
> recently purchased a new E.coli strain from Stratagene (their new BL21
> cells). Anyways, they recommend making up SOB medium, adding sterile
> glucose (then it becomes SOC medium, I think), and transforming their
> cells with SOC *and* B-mercaptoethanol.... talk about a pain in the neck!
> I've tried it once, and it is a long and tedious procedure -- and they
> say to make up the SOC fresh every time. I know this is what
> Stratagene recommends, but honestly, is this absolutely necessary?
> (I would prefer to use the "add plasmid, incubate on ice, heat shock, add
> LB, mix 1 hr, plate). So essentially, the question is: Does it make a
> difference for cell "health" (or further down the road, protein
> expression) depending on what protocol one uses?
> What would be the purpose of B-mercaptoethanol anyways?
there are several reasons for Stratagene to recommend this protocol.
BL21 bacteria (even the newer strains with improved transformability)
are difficult to transform, giving a yield of clones per ug of DNA that
is some 2 orders of magnitude lower than, eg, XL1blue bacteria. Thus,
all methods to raise the efficiency of transformation may be applied.
One possibility is to use a richer medium for the recovery period after
heat shock. SOC contains glucose and results in a higher number of
clones per ug of DNA because more cells survive the transformation.
The other possibility is to add low amounts of mercaptoethanol, as this
has shown to improve efficiency as well, possibly because it protects
sulfydryl groups in critical bacterial proteins. I think the exact
mechanism is not known. The effect, at least in my hands, is marginal,
possibly a factor of two.
Bottomline: When you transform bacteria in order to make protein (and
that is the only reason i can imagine one would use BL21), it does not
really matter. Use enough DNA to get a decent number of clones. That´s
it. It will take more DNA than you would usually use for, eg. XL1blue.
100ng is certainly a good starting point. Then you are also free to use
LB and leave out the beta-MSH.
NEVER use a ligation mix to transform BL21. It simply won´t work because
of the low efficiency of transformation with ligation products (which,
in all strains I know, is rougly 2-3 orders of magnitude lower than that
of supercoiled DNA). Miniprep DNA usually works well, we use 1-5ul of a
PS: If you need to transform BL21 with high efficiency, use
electroporation. It is also much faster than chemical transformation.
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