[HELP]Can't transformat linear plasmid into B. subtilis by electroporation!

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Mar 28 10:07:19 EST 2001


Hayes Chen <Beloved at ms26.url.com.tw> wrote:

:Hello, I am trying to transfrom a linear plasmid (~10kb) into B.
:subtilis by electroporation, and  BTX's ECM399 electroporation system is
:used, but the protocol provided by the agent is so ambiguous that I must
:GUESS what it means. Till now I am having doen electroporation many
:times with failing results !
:The lately protocol I used to prepare B. subtilis competent cell : B.
:subtilis 168  is cultured in LB, 37 degree overnight from a single
:colony, and reculture it with 100X dilution. When growing to OD600 1.5,
:centrifuge to collect pellet and  wash the pellet with dd-H2O three
:times, and finally suspend it with 1/100 culture volume of 30% PEG6000.
:Electroproation condition is 2500V, 5 secs applying on 0.5ug DNA/100ul
:cell mixture.
:Please anyone who have done electroporation with B. subtilis give some
:suggestion or provide a protocol to me.

The problem might be with plasmid being linear. For most
bacteria this form is non transforming. Quick search in 
medline gives this hit:
----------
Effects of DNA topology on transformation efficiency of Bacillus 
subtilis ISW1214 by electroporation. Bioscience, Biotechnology & 
Biochemistry.  61(6):1019-21, 1997

The  transformation with the sc DNA of pUB110 resulted in the 
maximum efficiency of (2.6 +/- 0.6) x 10(5) transformants per 
microgram DNA higher than that (2.0 +/- 0.3) x 10(4) transformants 
per microgram DNA for the cr DNA, using the DNA concentration of 20 
micrograms/ml at an electric field strength of 7 kV/cm and a 
capacitance of 10 microF with a single decayed pulse. The 
transformation efficiency (TE) for the ln DNA was zero.
-----

Do a control with supercoiled form. Also, you don't mention 
what cuvette you use. It's not the voltage that matters but it's 
drop, which depends on the distance between electrodes. 
Make sure to have everything ice-cold to prevent boiling 
bacteria. Unless PEG does something to the cell wall, 
I'd try to do away with PEG. 30% PEG-6000 is going to make
solute very viscosous, and this decreases electroporation 
efficiency by decreasing DNA electrophoretic movement.

        - Dima




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