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