TSS Transformation problems
s535290 at aix1.uottawa.ca
Fri May 30 16:24:59 EST 1997
I usually make separate stock solutions of everything. I autoclave a %40
PEG 4000 solution, filter sterilize my stock of DMSO (I don't even know if
this is necessary), and make sterile 2xLB with the MgCl2 already added.
Then all you have to do is mix up the different components and everything
should stay dissolved. Most of the time I don't use 2XTSS though and
spin down my cells and resuspend in 1XTSS because I seem to get
better transformation efficiencies (I also use a bit more cells than is
mentioned in the protocol - about 50% more cells). I harvest the
cells and resuspend the cell pellet in LB + MgCl2, and once the pellet is
fully resuspended, I go on and add first the DMSO and lastly the PEG.
Works like a charm, and I routinely get 1 to 5 x 10 E7 per microgram of
ccc pUC18. I make batches of 50 to 100 by flash freezing in liquid N2.
> I'm having a go at the quick and easy transformation method of Chung et
> al. as advertised in this newsgroup, but I'm concerned about making up the
> initial 2x TSS solution. When I mix 20% PEG 4000, 10% DMSO, and 100mM
> MgCl2 with my 2xLB I get a very murky looking mixture, not all of which is
> dissolved. Is this what you expect, and how am I supposed to sterilise
> it? Filter sterilisation gives a nice clear liquid, but I suspect it's
> taken out some of the ingredients.
> Thanks in advance for any help,
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