Optimal Electroporation settings for JM109
sbeisma at Uni-MolGen.gwdg.de
Mon Jul 20 10:32:01 EST 1998
Tam Anh Le wrote:
> Hello all
> I have always used heat shock as a methods of transforming bacterial
> cell with my desired plasmid, however my supervisor has told me that
> electroporation is a more effective than heat shock. To that extend I want
> to try this method of transformation, however I do not have the information
> regarding the optimal setting host strain JM109, I am hoping someone out has
> this information and could share it with me.
> -Many thanks
> Tam Anh Le
> Clinical Neuroscience
> St Vincent Hospital Melbourne.
Hi Tam Anh,
this is the way I carry out the electroporation:
fill 50 microliter electrocompetent E. colis (e. g. JM109)into a
procooled electroporation cuvette, add 2-4 microliter desalted
(dialysed) ligation product and mix gently. Insert into a gene pulser
(e.g. from BioRAD) and then:1 pulse at 200 Ohm, 25 microF, 2.5 kV.
If the DNA is readily desalted you will hear a buzzing sound- otherwise
a big bang! You can still try to propagate the survivers...
Add 1 ml of SOC immediately after the pulse and cure for 1 h at 37
degree. Plate 100 to 300 microliter on plates with appropriate medium
(e.g. with antibiotics). Thats it!
SOC: 2 percent trypton
0.5 percent yeast extract
10 mM NaCL
2.5 mM KCl#
20 mM MgCl2
then: add 20 mM glucose.
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