http://www.openwetware.org/index.php?title=Mathies:E_COLI_PCR&feed=atom&action=history 2013-05-19T14:01:20Z Revision history for this page on the wiki MediaWiki 1.13.2 http://www.openwetware.org/index.php?title=Mathies:E_COLI_PCR&diff=327093&oldid=prev 2009-07-23T05:22:24Z

<p>New page: <a href="/wiki/Category:Protocol" title="Category:Protocol">Category:Protocol</a> <a href="/wiki/Category:Microfluidics" title="Category:Microfluidics">Category:Microfluidics</a> &lt;!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --&gt; ==E. COLI PCR== '''Reagents''' Templates: E. Coli K12 or O157 Primers...</p> <p><b>New page</b></p><div>[[Category:Protocol]]<br /> [[Category:Microfluidics]]<br /> <br /> &lt;!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --&gt;<br /> <br /> <br /> ==E. COLI PCR==<br /> <br /> '''Reagents'''<br /> <br /> Templates: E. Coli K12 or O157<br /> <br /> Primers: Primers specific to the KI#128 island on the K12 genome (forward primer: 5' - TTC GAT TAC ACG GAG TGC TGG GAA - 3' and reverse primer: 5' - CGT TGA TTT GCC GTT CCA TGT CGT - 3'; 259 bp product) and the OI#43 island on the O157 genome (forward primer: 5' - TGG CAG GAA GAG AGT GAC AGG - 3' and reverse primer: 5' - GGC CTT ACC CGT GAA CAG TA - 3'; 191 bp product) were designed to prevent any cross-amplification between strains. Forward primers were labeled with a 6-FAM dye on the 5-prime end (Integrated DNA Technologies). (Lewis 313 F1 Bottom Left “Lina”) *bleep*tail: Qiagen HotStarTaq Master Mix (400 µM each dNTP, 3 mM MgCL2, 0.1 units/µL Taq)<br /> <br /> PCR-proof water<br /> <br /> ==Protocol==<br /> <br /> 1µl of each template<br /> <br /> 1µl 10µM of forward and reverse primers<br /> <br /> 12.5µl of Qiagen HotStarTaq Master Mix<br /> <br /> 10.5µl of water<br /> <br /> Thermal cycling program: SAPCR<br /> <br /> 10 minute 95 °C (hot start step to activate the Taq polymerase)<br /> <br /> 29 cycles of 94 °C denaturation (30 sec), 60 °C annealing (30 sec), and 72 °C extension (30 sec)<br /> <br /> 5 minute 72 °C<br /> <br /> Incubate at 4 °C before other analysis<br /> <br /> ==Contact==<br /> *'''[[User:Eric Chu|Eric Chu]] 22:25, 22 July 2009 (PDT)''':<br /> <br /> or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. <br /> <br /> &lt;!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --&gt;<br /> <br /> [[Category:Protocol]]<br /> [[Category:Microfluidics]]<br /> <br /> <br /> &lt;!-- Move the relevant categories above this line to tag your protocol with the label<br /> [[Category:Protocol]]<br /> <br /> [[Category:Needs attention]]<br /> <br /> [[Category:In vitro]]<br /> <br /> [[Category:In vivo]]<br /> <br /> [[Category:In silico]]<br /> <br /> [[Category:DNA]]<br /> <br /> [[Category:RNA]]<br /> <br /> [[Category:Protein]]<br /> <br /> [[Category:Chemical]]<br /> <br /> [[Category:Escherichia coli]]<br /> <br /> [[Category:Yeast]]<br /> --&gt;</div>

Eric Chu