Electroporators for bacterial transformation
num208jn at MBDS.NRC.CA
Fri Nov 8 11:11:02 EST 1991
In article <9111080914.AA01786 at genbank.bio.net>, PHILLIPSA at LARS.AFRC.AC.UK writes:
>I'm considering buying an electroporator for routine transformation
>of E.coli (XL1blue, DH5alpha etc) with plasmids and plasmid
>ligations, to try to increase the number of transformants from
>"difficult" cloning experiments. I need to know:
>1. Do these machines really produce the advertised yield per ug (eg.
> 1010) ie. are they worth having?
YES!! Although the yield also depends on the plasmid/phagemid. I can
EASILY get 10^10 with pUC19, but get less with anything with an ssORI
like pUC119 or BRL's pT7/T3alpha-19. Also if you put some inserts
into pUC, even though the control pUC gives 10^10, the insert doesn't.
Since this also happens in a similar fashion with chemical
transformation, I'm not at all surprised.
>2. How easy are they to use/optimise?
For E. coli, I found it easy --> just follow the instructions in the
manual that comes with the electroporator (in BioRad's case). I make
the electrocompetent cells once every three or so months and freeze
them. (It's basically make 1 l of log phase cells, wash acouple of
times in water, once in 10% glycerol, resuspend them in 2 ml 10%
glycerol, and freeze 20 - 40 ul samples. Then it's thaw, mix, zap,
incubate, dilute (!!) and plate.
One downside: the cuvettes aren't cheap and are not really usable..
from the top of my head, I think I pay something like $Cdn 2.50 -
$2.80 /cuvette. Also, I find that one or two per box arc, which makes
a loud noise and scares the $%^& out of me for a few minutes.
>3. Who sells the best (or, more importantly, best value)
We like our BioRad, and the lab up the hall likes their BRL. One
advantage of BRL's (I believe) is that you can do multiple samples per
zap, but we paid signicantly less for our BioRad (their list prices
are fairly close, so it depends on company promotions, I suppose).
>Is there anybody out there who can help me?
Hope I did...
>Thanks - Andy Phillips (EARN: PHILLIPSA at LARS.AFRC.AC.UK)
>AFRC-IACR Long Ashton Research Station
> Bristol BS18 9AF, UK
John Nash | Internet: Nash at biologysx.lan.nrc.ca
Institute for Biological Sciences | or: num208jn at mbds.nrc.ca
National Research Council of Canada| Ottawa, Canada K1A 0R6.
==> Disclaimer: All opinions are mine, not NRC's! <==
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