http://2009.igem.org/wiki/index.php?title=Team:IIT_Bombay_India/protocol&feed=atom&action=history
Team:IIT Bombay India/protocol - Revision history
2024-03-28T23:47:59Z
Revision history for this page on the wiki
MediaWiki 1.16.5
http://2009.igem.org/wiki/index.php?title=Team:IIT_Bombay_India/protocol&diff=156598&oldid=prev
Pranayiitb at 21:55, 21 October 2009
2009-10-21T21:55:03Z
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<td colspan='2' style="background-color: white; color:black;">Revision as of 21:55, 21 October 2009</td>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>0.1M potassium phosphate buffer</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">'''</ins>0.1M potassium phosphate buffer<ins class="diffchange diffchange-inline">'''</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </div></td></tr>
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Pranayiitb
http://2009.igem.org/wiki/index.php?title=Team:IIT_Bombay_India/protocol&diff=156585&oldid=prev
Pranayiitb at 21:54, 21 October 2009
2009-10-21T21:54:32Z
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Z BUFFER'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Z BUFFER'''</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">''</ins>(60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)<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>(60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; 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>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 40 mg ONPG </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 40 mg ONPG </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 class="diffchange diffchange-inline">[[Media:Example.ogg]]</del>10.0 ml 0.1 potassum phosphate buffer pH7.0 </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• 10.0 ml 0.1 potassum phosphate buffer pH7.0 </div></td></tr>
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Pranayiitb
http://2009.igem.org/wiki/index.php?title=Team:IIT_Bombay_India/protocol&diff=156544&oldid=prev
Pranayiitb at 21:53, 21 October 2009
2009-10-21T21:53:02Z
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<tr><td colspan="2"> </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;">{| style="color:#000000;background-color:#ffffff;" cellpadding="6" cellspacing="3" border="0" bordercolor="#ffffff" width="65%" align="center"</ins></div></td></tr>
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<tr><td colspan="2"> </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;">!align="center"|[[Team:IIT_Bombay_India|Home]]</ins></div></td></tr>
<tr><td colspan="2"> </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;">!align="center"|[[Team:IIT_Bombay_India/Team|The Team]]</ins></div></td></tr>
<tr><td colspan="2"> </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;">!align="center"|[[Team:IIT_Bombay_India/Project|The Project]]</ins></div></td></tr>
<tr><td colspan="2"> </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;">!align="center"|[[Team:IIT_Bombay_India/Analysis|Analysis]]</ins></div></td></tr>
<tr><td colspan="2"> </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;">!align="center"|[[Team:IIT_Bombay_India/Modeling|Modeling]]</ins></div></td></tr>
<tr><td colspan="2"> </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;">!align="center"|[[Team:IIT_Bombay_India/Notebook|Notebook]]</ins></div></td></tr>
<tr><td colspan="2"> </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;">!align="center"|[[Team:IIT_Bombay_India/Safety|Safety]]</ins></div></td></tr>
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<tr><td colspan="2"> </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;">[[Image:IITB-Home.jpg]]</ins></div></td></tr>
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<tr><td colspan="2"> </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;">!align="left"|</ins></div></td></tr>
<tr><td colspan="2"> </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"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Competent cell preparation of E.coli Strain MG1655(lacI deleted)'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Competent cell preparation of E.coli Strain MG1655(lacI deleted)'''</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </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>• 6.0ml 1M Na2HPO4 stock solution</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• <ins class="diffchange diffchange-inline"> </ins>6.0ml 1M Na2HPO4 stock solution</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: #ffa; color:black; font-size: smaller;"><div>• 4.0 ml NaH2PO4 stock solution </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• <ins class="diffchange diffchange-inline"> </ins>4.0 ml NaH2PO4 stock solution </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>• 0.33 ml 3M KCL stock solution </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• <ins class="diffchange diffchange-inline"> </ins>0.33 ml 3M KCL stock solution </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>• 1.0ml 100mM MgSO4 stock solution</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• <ins class="diffchange diffchange-inline"> </ins>1.0ml 100mM MgSO4 stock solution</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>• 350 ul 2-mercaptoethanol </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• <ins class="diffchange diffchange-inline"> </ins>350 ul 2-mercaptoethanol </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>Bring volume to 100 ml with distilled H2O. Do not autoclave!</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Bring volume to 100 ml with distilled H2O. Do not autoclave!</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 10ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 10ml: </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>• 40 mg ONPG </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• <ins class="diffchange diffchange-inline"> </ins>40 mg ONPG </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>• 10.0 ml 0.1 potassum phosphate buffer pH7.0 </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• <ins class="diffchange diffchange-inline"> [[Media:Example.ogg]]</ins>10.0 ml 0.1 potassum phosphate buffer pH7.0 </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>
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Pranayiitb
http://2009.igem.org/wiki/index.php?title=Team:IIT_Bombay_India/protocol&diff=156506&oldid=prev
Pranayiitb at 21:51, 21 October 2009
2009-10-21T21:51:41Z
<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 21:51, 21 October 2009</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Transformation Of MG1655'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Transformation Of MG1655'''</div></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;"></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Thaw 200ul of competent cells on ice properly.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Thaw 200ul of competent cells on ice properly.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Growth curve'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Growth curve'''</div></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;"></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Single colony was inoculated into 5ml LB with appropriate antibiotic.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Single colony was inoculated into 5ml LB with appropriate antibiotic.</div></td></tr>
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<td colspan="2" class="diff-lineno">Line 69:</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>'''Microscopy'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Microscopy'''</div></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;"></del></div></td><td colspan="2"> </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>1.Around 5ul of the culture was placed on a thin glass microscopic slide. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1.Around 5ul of the culture was placed on a thin glass microscopic slide. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Fluorescence activated Cell sorting'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Fluorescence activated Cell sorting'''</div></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;"></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1.Cells were grown for required time (for dynamics) or to a particular O.D. on M9 medium.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1.Cells were grown for required time (for dynamics) or to a particular O.D. on M9 medium.</div></td></tr>
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<td colspan="2" class="diff-lineno">Line 101:</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>'''Beta galactosidase assay'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Beta galactosidase assay'''</div></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;"></del></div></td><td colspan="2"> </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>1. Grow 5ml LB cultures to mid-log phase.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Grow 5ml LB cultures to mid-log phase.</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 142:</td>
<td colspan="2" class="diff-lineno">Line 137:</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>'''Z BUFFER'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Z BUFFER'''</div></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;"></del></div></td><td colspan="2"> </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>(60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 148:</td>
<td colspan="2" class="diff-lineno">Line 142:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </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>• 6.0ml 1M Na2HPO4 stock solution </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>• 6.0ml 1M Na2HPO4 stock solution</div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 4.0 ml NaH2PO4 stock solution </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 4.0 ml NaH2PO4 stock solution </div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 0.33 ml 3M KCL stock solution </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 0.33 ml 3M KCL stock solution </div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 1.0ml 100mM MgSO4 stock solution</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 1.0ml 100mM MgSO4 stock solution</div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 350 ul 2-mercaptoethanol </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 350 ul 2-mercaptoethanol </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 colspan="2" class="diff-lineno">Line 159:</td>
<td colspan="2" class="diff-lineno">Line 157:</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>'''ONPG'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''ONPG'''</div></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;"></del></div></td><td colspan="2"> </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>To make 10ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 10ml: </div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 40 mg ONPG </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 40 mg ONPG </div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 10.0 ml 0.1 potassum phosphate buffer pH7.0 </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 10.0 ml 0.1 potassum phosphate buffer pH7.0 </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 colspan="2" class="diff-lineno">Line 169:</td>
<td colspan="2" class="diff-lineno">Line 168:</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>To make 100ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• Make solution A: 27.2 g KH2PO4 in 1 L water.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• Make solution A: 27.2 g KH2PO4 in 1 L water.</div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• Make solution B: 34.8 g K2HPO4 in 1 L water.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• Make solution B: 34.8 g K2HPO4 in 1 L water.</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>
</table>
Pranayiitb
http://2009.igem.org/wiki/index.php?title=Team:IIT_Bombay_India/protocol&diff=156481&oldid=prev
Pranayiitb at 21:50, 21 October 2009
2009-10-21T21:50:25Z
<p></p>
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 21:50, 21 October 2009</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Competent cell preparation of E.coli Strain MG1655(lacI deleted)'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Competent cell preparation of E.coli Strain MG1655(lacI deleted)'''</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>
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<td colspan="2" class="diff-lineno">Line 30:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>14. Aliquotes in eppendorf tubes (ice cold) to a volume of 200ul each.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>14. Aliquotes in eppendorf tubes (ice cold) to a volume of 200ul each.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Transformation Of MG1655'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Transformation Of MG1655'''</div></td></tr>
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<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 colspan="2" class="diff-lineno">Line 49:</td>
<td colspan="2" class="diff-lineno">Line 51:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. keep the plates at 37 degree in incubator.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. keep the plates at 37 degree in incubator.</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>'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Growth curve'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Growth curve'''</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; 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>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Single colony was inoculated into 5ml LB with appropriate antibiotic.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Single colony was inoculated into 5ml LB with appropriate antibiotic.</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 63:</td>
<td colspan="2" class="diff-lineno">Line 67:</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>6. O.D. was plotted against time on Excel sheet and Specific growth rate was calculated from the exponential phase.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>6. O.D. was plotted against time on Excel sheet and Specific growth rate was calculated from the exponential phase.</div></td></tr>
<tr><td colspan="2"> </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 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;"></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>'''Microscopy'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Microscopy'''</div></td></tr>
<tr><td colspan="2"> </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 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>1.Around 5ul of the culture was placed on a thin glass microscopic slide. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1.Around 5ul of the culture was placed on a thin glass microscopic slide. </div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 76:</td>
<td colspan="2" class="diff-lineno">Line 82:</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>5.Image was captured.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>5.Image was captured.</div></td></tr>
<tr><td colspan="2"> </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 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;"></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>'''Fluorescence activated Cell sorting'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Fluorescence activated Cell sorting'''</div></td></tr>
<tr><td colspan="2"> </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 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>1.Cells were grown for required time (for dynamics) or to a particular O.D. on M9 medium.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1.Cells were grown for required time (for dynamics) or to a particular O.D. on M9 medium.</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 93:</td>
<td colspan="2" class="diff-lineno">Line 101:</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>7.The Data was analysed and its statistical value was calculated with the help of BD FACSARIA software.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>7.The Data was analysed and its statistical value was calculated with the help of BD FACSARIA software.</div></td></tr>
<tr><td colspan="2"> </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 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;"></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>'''Beta galactosidase assay'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Beta galactosidase assay'''</div></td></tr>
<tr><td colspan="2"> </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 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>1. Grow 5ml LB cultures to mid-log phase.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Grow 5ml LB cultures to mid-log phase.</div></td></tr>
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<td colspan="2" class="diff-lineno">Line 138:</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>Where Vol is volume of culture used in assay in mls, and Time is minutes at 30 degree C.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Where Vol is volume of culture used in assay in mls, and Time is minutes at 30 degree C.</div></td></tr>
<tr><td colspan="2"> </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"> </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 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>'''Z BUFFER'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Z BUFFER'''</div></td></tr>
<tr><td colspan="2"> </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"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>(60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)</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>To make 100ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 100ml: </div></td></tr>
<tr><td colspan="2"> </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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 6.0ml 1M Na2HPO4 stock solution </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 6.0ml 1M Na2HPO4 stock solution </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 4.0 ml NaH2PO4 stock solution </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 4.0 ml NaH2PO4 stock solution </div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 140:</td>
<td colspan="2" class="diff-lineno">Line 155:</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>Bring volume to 100 ml with distilled H2O. Do not autoclave!</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Bring volume to 100 ml with distilled H2O. Do not autoclave!</div></td></tr>
<tr><td colspan="2"> </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 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;"></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>'''ONPG'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''ONPG'''</div></td></tr>
<tr><td colspan="2"> </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 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>To make 10ml: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To make 10ml: </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 40 mg ONPG </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 40 mg ONPG </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 10.0 ml 0.1 potassum phosphate buffer pH7.0 </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>• 10.0 ml 0.1 potassum phosphate buffer pH7.0 </div></td></tr>
<tr><td colspan="2"> </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 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>0.1M potassium phosphate buffer</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>0.1M potassium phosphate buffer</div></td></tr>
</table>
Pranayiitb
http://2009.igem.org/wiki/index.php?title=Team:IIT_Bombay_India/protocol&diff=156426&oldid=prev
Pranayiitb: New page: '''Competent cell preparation of E.coli Strain MG1655(lacI deleted)''' 1.Take an overnight grown saturated E.coli cells in 5 ml LB and inoculate about 1/100 dilution in 50 ml culture. 2...
2009-10-21T21:48:21Z
<p>New page: '''Competent cell preparation of E.coli Strain MG1655(lacI deleted)''' 1.Take an overnight grown saturated E.coli cells in 5 ml LB and inoculate about 1/100 dilution in 50 ml culture. 2...</p>
<p><b>New page</b></p><div>'''Competent cell preparation of E.coli Strain MG1655(lacI deleted)'''<br />
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1.Take an overnight grown saturated E.coli cells in 5 ml LB and inoculate about 1/100 dilution in 50 ml culture.<br />
<br />
2. Grow it at 37 degree Celsius till the O.D. reaches upto 0.5 to 0.8.<br />
<br />
3. Transfer the culture to Cold autoclave round bottom centrifuge tube . Then keep them on ice for 10 minutes.<br />
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4.Spin at 5000 rpm for 10 minutes at 4 degree Celsius.<br />
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5.Decant the Supernatant .<br />
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6. keep the tube in upside down for 5 minutes on tissue paper pad to remove the residual medium.<br />
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7. Resuspend the pellet in 1/10 volume .1 M Cacl2 .<br />
<br />
8. Keep on ice for 40 minutes.<br />
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9. Spin at 5000 rpm for 10 minutes at 4 degree Celsius.<br />
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10. Discard the supernatant.<br />
<br />
11. Resuspend the entire pellet in 1/25 volume of the .1 M cacl2.<br />
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12. Keep on ice for 60 minutes.<br />
<br />
13. Add 15% glycerol (ice cold and autoclaved) to suspended cells.<br />
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14. Aliquotes in eppendorf tubes (ice cold) to a volume of 200ul each.<br />
<br />
'''<br />
Transformation Of MG1655'''<br />
<br />
<br />
1. Thaw 200ul of competent cells on ice properly.<br />
<br />
2. Add around 100 nano gram of plasmid.<br />
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3. Keep on ice for 30 minutes.<br />
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4. Give heat shock at 42 degree Celsius for 90 sec to 120 minutes.<br />
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5. keep on ice for 10 minutes.<br />
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6. keep the tube at 37 degree Celsius for around half an hour.<br />
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7. Spread around 100ul on the appropriate selection LB plates.<br />
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8. keep the plates at 37 degree in incubator.<br />
<br />
'''<br />
Growth curve'''<br />
<br />
1. Single colony was inoculated into 5ml LB with appropriate antibiotic.<br />
<br />
2. It was allowed to grow overnight.<br />
<br />
3. The culture O.D . was checked .<br />
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4. It was added to 50 ml LB /Defined media to a volume of 1/100.<br />
<br />
5. Every one hour the O.D. was checked at 600 nm.<br />
<br />
6. O.D. was plotted against time on Excel sheet and Specific growth rate was calculated from the exponential phase.<br />
<br />
<br />
'''Microscopy'''<br />
<br />
1.Around 5ul of the culture was placed on a thin glass microscopic slide. <br />
<br />
2.Mounted with coverslip.<br />
<br />
3.Focused.<br />
<br />
4.Exposed to blue laser.<br />
<br />
5.Image was captured.<br />
<br />
<br />
'''Fluorescence activated Cell sorting'''<br />
<br />
1.Cells were grown for required time (for dynamics) or to a particular O.D. on M9 medium.<br />
<br />
2.cells were pelleted at 4500 rpm for 10 minutes at room temperature.<br />
<br />
3.Cells were washed twice in 1X PBS.<br />
<br />
4.Cells were taken to a density of around 10 8 per ml.<br />
<br />
5.The samples were exposed to blue laser for YFP and violet laser for CFP.<br />
<br />
6. 5000 events were scored.<br />
<br />
7.The Data was analysed and its statistical value was calculated with the help of BD FACSARIA software.<br />
<br />
<br />
'''Beta galactosidase assay'''<br />
<br />
1. Grow 5ml LB cultures to mid-log phase.<br />
<br />
2. Centrifuge and resuspend cells in 5ml Z-buffer, then place on ice.<br />
<br />
3. Measure O.D.(600).<br />
<br />
4. Use straight, or dilute cell mix 10x or 20x (40 or 80ul brought to 0.8ml with Z-buffer).<br />
<br />
5. Using Pasteur pipet, add 1 drop of 0.1% SDS and 2 drops of chloroform to each tube.<br />
<br />
6. Vortex well for 15 seconds.<br />
<br />
7. Equilibrate at 30oC for 15 minutes.<br />
<br />
8. Add 160ul of 4mg/ml ONPG, and vortex well for 10 sec.<br />
<br />
9. Incubate at 30oC and begin timing.<br />
<br />
10. Remove after about 15-20 minutes (empirically determined by color).<br />
<br />
11. Quench reaction by adding 400 ul of 1M sodium carbonate.<br />
<br />
12. Spin down cell debris.<br />
<br />
13. Measure O.D.(420) and O.D.(550).<br />
<br />
14. Calculate Units using the following formula:<br />
<br />
U= 1000 x [(OD420)-(1.75 x OD550)] / [(Time) x (Vol) x OD600]<br />
<br />
Where Vol is volume of culture used in assay in mls, and Time is minutes at 30 degree C.<br />
<br />
'''Z BUFFER'''<br />
(60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)<br />
<br />
To make 100ml: <br />
• 6.0ml 1M Na2HPO4 stock solution <br />
• 4.0 ml NaH2PO4 stock solution <br />
• 0.33 ml 3M KCL stock solution <br />
• 1.0ml 100mM MgSO4 stock solution<br />
• 350 ul 2-mercaptoethanol <br />
<br />
Bring volume to 100 ml with distilled H2O. Do not autoclave!<br />
<br />
<br />
'''ONPG'''<br />
<br />
To make 10ml: <br />
• 40 mg ONPG <br />
• 10.0 ml 0.1 potassum phosphate buffer pH7.0 <br />
<br />
0.1M potassium phosphate buffer<br />
<br />
To make 100ml: <br />
• Make solution A: 27.2 g KH2PO4 in 1 L water.<br />
• Make solution B: 34.8 g K2HPO4 in 1 L water.<br />
<br />
Mix 39 ml Solution A and 61 ml Solution B and then add 100 ml of water.<br />
<br />
<br />
<br />
<br />
<br />
<br />
________________________________________</div>
Pranayiitb