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<DIV><FONT face=Arial size=2>In response to Rene's question about transforming
B. stearothermophilus - I had big problems</FONT></DIV>
<DIV><FONT face=Arial size=2>transforming this bug a few years ago (and in fact
appealed for help in this newsgroup and recieved some very helpful
replies).</FONT></DIV>
<DIV> </DIV>
<DIV><FONT face=Arial size=2>Anyway, I found the method that worked best was
Issay Narumi's electroporation method (published in</FONT></DIV>
<DIV><FONT face=Arial size=2>Biotechnology Letters, around 1993, I think). There
is great variation in efficiency of electrotransformation between
different</FONT></DIV>
<DIV><FONT face=Arial size=2>strains of B. stearothermophilus - it worked very
well with Dr. Narumi's strain K1041. In fact it turns out that this
strain</FONT></DIV>
<DIV><FONT face=Arial size=2>is actually Bacillus thermodenitrificans rather
than B. stearothermophilus - but this may or may not not be a problem for
you.</FONT></DIV>
<DIV> </DIV>
<DIV><FONT face=Arial size=2>As a positive control for the transformation DNA
you can use pUB110 DNA which you can buy from Sigma-Aldrich. This
confers</FONT></DIV>
<DIV><FONT face=Arial size=2>Kanamycin resistance. </FONT><FONT face=Arial
size=2>Also the methylation state of the DNA seems to be important- so be
careful if you use any shuttle vectors that have been passged through E.
coli.</FONT></DIV>
<DIV> </DIV>
<DIV><FONT face=Arial size=2>Good luck!</FONT></DIV>
<DIV> </DIV>
<DIV><FONT face=Arial size=2>Dave</FONT></DIV>
<DIV> </DIV>
<DIV> </DIV>
<DIV><FONT face=Arial
size=2>============================================<BR>David J. Studholme,
PhD.</FONT></DIV>
<DIV> </DIV>
<DIV><FONT face=Arial size=2>Division of Biochemistry & Molecular
Biology<BR>School of Biological Sciences<BR>University of Southampton<BR>Bassett
Crescent East<BR>SOUTHAMPTON<BR>SO16 7PX<BR>UK</FONT></DIV>
<DIV> </DIV>
<DIV><FONT face=Arial size=2><A
href="mailto:djs@soton.ac.uk">djs@soton.ac.uk</A><BR><A
href="http://promscan.uklinux.net">http://promscan.uklinux.net</A><BR>============================================
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