http://www.openwetware.org/index.php?title=Lidstrom:Gibson_Assembly&feed=atom&action=history 2013-06-19T07:17:06Z Revision history for this page on the wiki MediaWiki 1.13.2 http://www.openwetware.org/index.php?title=Lidstrom:Gibson_Assembly&diff=684988&oldid=prev 2013-03-22T20:21:39Z

<p><span class="autocomment">Transformation</span></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 20:21, 22 March 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 18:</td> <td colspan="2" class="diff-lineno">Line 18:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Electroporation can give much higher efficiency.&nbsp; For this reason, the assembled product is usually electroporated.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Electroporation can give much higher efficiency.&nbsp; For this reason, the assembled product is usually electroporated.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**rxn product is salty: can be a problem for electrocompetent cells.&nbsp; Rob Egbert dilutes the competent cells when transforming with electroporation.&nbsp; Not so much a problem when using chemically competent cells.&nbsp; &nbsp;</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**rxn product is salty: can be a problem for electrocompetent cells.&nbsp; Rob Egbert dilutes the competent cells when transforming with electroporation.&nbsp; Not so much a problem when using chemically competent cells.&nbsp; &nbsp;</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>** Janet <del class="diffchange diffchange-inline">hasn't had a problem transforming 1 </del>uL <del class="diffchange diffchange-inline">of product if a cuvette is new.&nbsp; Older cuvettes arc more easily. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>** Janet <ins class="diffchange diffchange-inline">electroporates 2 </ins>uL</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*</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> <!-- diff generator: internal 2013-06-19 07:17:06 --> </table>

Janet B. Matsen http://www.openwetware.org/index.php?title=Lidstrom:Gibson_Assembly&diff=684984&oldid=prev 2013-03-22T20:15:01Z

<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 20:15, 22 March 2013</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 1:</td> <td colspan="2" class="diff-lineno">Line 1:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Back to [[Lidstrom:Protocols]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Back to [[Lidstrom:Protocols]]</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;"></ins></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;">== Janet's Tips == </ins></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;">'''Janet is developing a protocol [[Janet B. Matsen:Guide to Gibson Assembly |here]]'''</ins></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: #eee; color:black; font-size: smaller;"><div>==Design:==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Design:==</div></td></tr> <!-- diff generator: internal 2013-06-19 07:17:06 --> </table>

Janet B. Matsen http://www.openwetware.org/index.php?title=Lidstrom:Gibson_Assembly&diff=631445&oldid=prev 2012-10-02T14:15:46Z

<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 14:15, 2 October 2012</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 1:</td> <td colspan="2" class="diff-lineno">Line 1:</td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Design:</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Back to [[Lidstrom:Protocols]]</ins></div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">- </del>overlap by 40 bp</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>&#160;</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 class="diffchange diffchange-inline">==</ins>Design:<ins class="diffchange diffchange-inline">==</ins></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 class="diffchange diffchange-inline">===Fragments===</ins></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 class="diffchange diffchange-inline">*</ins>overlap by 40 bp</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 class="diffchange diffchange-inline">*efficiency </ins></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 class="diffchange diffchange-inline">===Oligo (primer) Design===</ins></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 class="diffchange diffchange-inline">*</ins></div></td></tr> <tr><td colspan="2">&nbsp;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>&#160;</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 class="diffchange diffchange-inline">==PCR==</ins></div></td></tr> <tr><td colspan="2">&nbsp;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>&#160;</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 class="diffchange diffchange-inline">==Assembly==</ins></div></td></tr> <tr><td colspan="2">&nbsp;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>&#160;</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 class="diffchange diffchange-inline">== Transformation==</ins></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 class="diffchange diffchange-inline">*Electroporation can give much higher efficiency.&nbsp; For this reason, the assembled product is usually electroporated.</ins></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 class="diffchange diffchange-inline">**rxn product is salty: can be a problem for electrocompetent cells.&nbsp; Rob Egbert dilutes the competent cells when transforming with electroporation.&nbsp; Not so much a problem when using chemically competent cells.&nbsp; </ins></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 class="diffchange diffchange-inline">** Janet hasn't had a problem transforming 1 uL of product if a cuvette is new.&nbsp; Older cuvettes arc more easily. </ins></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 class="diffchange diffchange-inline">*</ins></div></td></tr> <tr><td colspan="2">&nbsp;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>&#160;</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 class="diffchange diffchange-inline">==Screening==</ins></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 class="diffchange diffchange-inline">* Colony PCR 1st</ins></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 class="diffchange diffchange-inline">* Sequence a few clones with primers that cover the &quot;seams.&quot;</ins></div></td></tr> <tr><td colspan="2">&nbsp;</td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>&#160;</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 class="diffchange diffchange-inline">==Misc: ==</ins></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><del style="color: red; font-weight: bold; text-decoration: none;">Misc:</del></div></td><td colspan="2">&nbsp;</td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">- rxn product is salty: can be a problem for electrocompetent cells.&nbsp; Rob Egbert dilutes the competent cells when transforming with electroporation.&nbsp; Not so much a problem when using chemically competent cells.&nbsp; </del></div></td><td colspan="2">&nbsp;</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>- Electrocompetence is ~ 1000x more effective so is tempting.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>- Electrocompetence is ~ 1000x more effective so is tempting.</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>-</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>-</div></td></tr> <!-- diff generator: internal 2013-06-19 07:17:06 --> </table>

Janet B. Matsen http://www.openwetware.org/index.php?title=Lidstrom:Gibson_Assembly&diff=587436&oldid=prev 2012-02-24T14:39:04Z

<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 14:39, 24 February 2012</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 1:</td> <td colspan="2" class="diff-lineno">Line 1:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Design:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Design:</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>- overlap by <del class="diffchange diffchange-inline">20 </del>bp</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>- overlap by <ins class="diffchange diffchange-inline">40 </ins>bp</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: #eee; color:black; font-size: smaller;"><div>Misc:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Misc:</div></td></tr> <!-- diff generator: internal 2013-06-19 07:17:06 --> </table>

Janet B. Matsen http://www.openwetware.org/index.php?title=Lidstrom:Gibson_Assembly&diff=587435&oldid=prev 2012-02-24T14:38:44Z

<p>New page: Design: - overlap by 20 bp Misc: - rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not ...</p> <p><b>New page</b></p><div>Design:<br /> - overlap by 20 bp<br /> <br /> Misc:<br /> - rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells. <br /> - Electrocompetence is ~ 1000x more effective so is tempting.<br /> -</div>

Janet B. Matsen