http://openwetware.org/index.php?title=Belcher/Knight:_Electrocompetent_Cells&feed=atom&action=history 2013-05-21T19:52:07Z Revision history for this page on the wiki MediaWiki 1.13.2 http://openwetware.org/index.php?title=Belcher/Knight:_Electrocompetent_Cells&diff=115499&oldid=prev 2007-05-08T21:15:25Z

<p></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 21:15, 8 May 2007</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 38:</td> <td colspan="2" class="diff-lineno">Line 38:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Category:Protocol]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Category:Protocol]]</div></td></tr> <tr><td colspan="2">&nbsp;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[Category:In vivo]]</ins></div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Category:Escherichia coli]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Category:Escherichia coli]]</div></td></tr> <!-- diff generator: internal 2013-05-21 19:52:07 --> </table>

Reshma P. Shetty http://openwetware.org/index.php?title=Belcher/Knight:_Electrocompetent_Cells&diff=90436&oldid=prev 2006-12-05T22:44:42Z

<p></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 22:44, 5 December 2006</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 37:</td> <td colspan="2" class="diff-lineno">Line 37:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For transformation usually use 40-50uL of competent cells per transformation with a few ul (2-3) of ligation.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For transformation usually use 40-50uL of competent cells per transformation with a few ul (2-3) of ligation.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>&#160;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Category:Protocol<ins class="diffchange diffchange-inline">]]</ins></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Category:<del class="diffchange diffchange-inline">E.Coli </del>Protocol]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">[[Category:Escherichia coli</ins>]]</div></td></tr> <!-- diff generator: internal 2013-05-21 19:52:07 --> </table>

Reshma P. Shetty http://openwetware.org/index.php?title=Belcher/Knight:_Electrocompetent_Cells&diff=78526&oldid=prev 2006-10-11T15:29:43Z

<p></p> <p><b>New page</b></p><div>==Electrocompetent Cells==<br /> <br /> <br /> From Danijela Dukovski at Harvard Medical School. This protocol works well.<br /> --Julie Norville<br /> <br /> ===Materials===<br /> DI water&lt;br&gt; <br /> 10% Glycerol&lt;br&gt;<br /> <br /> ===Special Equipment===<br /> Centrifuge&lt;br&gt;<br /> Ice water bath&lt;br&gt;<br /> Liquid nitrogen&lt;br&gt;<br /> <br /> ===Method===<br /> Grow 500ml culture to OD 0.5 (approximately).<br /> <br /> Spin down cells 5 times in ice cold 10% sterile glycerol.<br /> <br /> Keep everything on ice and use a refrigerated centrifuge.<br /> <br /> Each time you resuspend use a progressively smaller volume, and make sure all the cells are well resuspended. At the end resuspend in an appropriate volume. It should be pretty cloudy but not super dense. I just do it by eye, so it comes out about like the competent cells you buy. <br /> <br /> Aliquot into Eppendorf tubes (I use 250-500uL, depending on how much I have and how lazy I am), freeze in liquid nitrogen, and store at -80.<br /> <br /> How I usually do the washes:<br /> spin, resuspend in 250ml<br /> spin, resuspend in 100ml<br /> spin, resuspend in 50ml (can switch to Falcon tubes here)<br /> spin, resuspend in 25ml<br /> spin, resuspend in 10ml<br /> spin, final resuspension<br /> <br /> Sometimes I cut one of these out depending on how much of a hurry I am in. I think the final volume (depends on the intial OD of your cells) usually ends up being 5-10mL, so you can get quite a few aliquots.<br /> <br /> For transformation usually use 40-50uL of competent cells per transformation with a few ul (2-3) of ligation.<br /> <br /> <br /> [[Category:E.Coli Protocol]]</div>

Norville