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Electroporation of M. tuberculosis - (Apr/09/2005 )

Hi everybody,
Could someone please please tell me how to increase the efficiency of electroporation of Mycobacterium tuberculosis. I sometimes find no colonies even after 3-4 months of incubation. Its very depressing.
100microlitre competent cells are electroporated with approximately 1-2microgram of plasmid DNA (7kb). Electroporation conditions are Voltage of 2.5kV, Capacitance of 25microF and resistance of 700ohms (I have tried 600-1000ohms) in a 0.2cm cuvette. The apparatus used is a Bio-Rad Gene Pulser II.
Thanking you in advance,
Madhavi.

-madhavilatha-

Madhavi,

Though I have not worked with the specific bacteria, I feel you are using too much of your plasmid! Normal electroporation works great at 2-3 ng of DNA. Also, try 1.5 kv, 25 uF, 200 ohms. I use only 20-40 ul of competent bacteria (home made DH5a, probably an easy one to electroporate!) Not sure whether this works for the bacteria that you are using.
Good luck.

SV

-SVTX-

QUOTE (SVTX @ Apr 10 2005, 05:50 PM)
Madhavi,

Though I have not worked with the specific bacteria, I feel you are using too much of your plasmid!  Normal electroporation works great at 2-3 ng of DNA.  Also, try 1.5 kv, 25 uF, 200 ohms. I use only 20-40 ul of competent bacteria (home made  DH5a, probably an easy one to electroporate!)  Not sure whether this works for the bacteria that you are using. 
Good luck.

SV


Thank you very much SV for your reply.
The conditions you have mentioned are actually used for M.smegmatis (a non pathogenic and fast growing bacteria of the genus). But M. tuberculosis is too tough for such milder conditions.
Still maybe I should try it that way too. At present I am ready to try anything to put the plasmid into the bacteria.
Madhavi.

-madhavilatha-