[HELP]Can't transformat linear plasmid into B. subtilis by
Beloved at ms26.url.com.tw
Tue Mar 27 22:12:44 EST 2001
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.
Thanks for any insight,
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