Basic Subcloning Question
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Mon Jun 7 06:45:38 EST 1999
In article <37595FC6.62CE942F at ibm.net>, Nicolas Lemee <Lemee at ibm.net>
> Again at 135$ a bag of
>Biorad cuvettes (not to mention the electroporator itself), I'd rather do heat
>shock and buy top quality cells.
Of course you could save all the money and make your own electroporator.
Straightforward to do as per a very old Biotechniques article (don't ask
me the year or author!) using fairly low voltage and very small gaps.
Mine uses 340V (240V AC rectified via an isolation transformer). Charge
a 22uf capacitor and discharge that across a gap created using two
appropriately insulated large aluminium blocks with a gap created by a
few layers of high voltage insulating tape (i.e. equivalent of
12500V/cm). By initially adjusting the gap and the capacitance one can
get very close to commercial machines for E.coli. You need cells in 20%
glycerol rather than 10%. Zap, overlay with 1ml SOC and remove cells. To
sterilize wipe the blocks with 70% EtOH and use again and again. Polish
as is required every 20 or so zaps (high voltage side only) with
automotive chrome cleaner.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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