http://www.openwetware.org/index.php?title=Lidstrom:Chemical_Transformation&feed=atom&action=history 2013-05-18T21:56:51Z Revision history for this page on the wiki MediaWiki 1.13.2 http://www.openwetware.org/index.php?title=Lidstrom:Chemical_Transformation&diff=629270&oldid=prev 2012-09-24T20:43:22Z

<p>New page: Back to <a href="/wiki/Lidstrom:Protocols" title="Lidstrom:Protocols">Protocols</a> You can chose between chemically competent cells and electrocompetent cells. Andrew &amp; Nicole make chemically competent cells for the lab to use. ...</p> <p><b>New page</b></p><div>Back to [[Lidstrom:Protocols|Protocols]]<br /> <br /> You can chose between chemically competent cells and electrocompetent cells. Andrew &amp; Nicole make chemically competent cells for the lab to use. Several strains are kept in stock in the -80oC freezer. <br /> <br /> ==Using Chemically Competent Cells==<br /> *Thaw frozen (-80oC) competent cells on ice.<br /> **You REALLY dont want it to get too warm; add plasmids while it is still slushy. <br /> *Add 1-10 uL DNA<br /> **10 uL if ligated plasmid. Use only 1 uL if regular plasmid from miniprep<br /> *Incubate @ 42oC for 45 sec - 1 min<br /> *Incubate on ice for 2 min<br /> *Add 1 mL LB <br /> **Sandy, Ceci, &amp; Janet use 500 uL<br /> *Incubate at 37oC for 45 min - 1 hr in eppendorf tubes<br /> **Ideally shaking though it may not matter. You can tape your tubes to a rack in the shaker. Tape them well if you do -- they fly off!<br /> *Pellet cells by centrifugation<br /> **keep the ~100 uL droplet after you pour it off (Andrew) <br /> *Plate 50-100 uL cells on LB (+antibiotic(s)) agar plate<br /> **If you are worried about having a lawn, do one plate with more cells and dilute a fraction of the cells and plate a diluted aliquot.</div>

Janet B. Matsen