http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_July_2009&feed=atom&action=historyTeam:Groningen/Notebook/8 July 2009 - Revision history2024-03-29T10:59:59ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_July_2009&diff=47961&oldid=prevFrans: /* Dry */2009-08-17T11:47:55Z<p><span class="autocomment">Dry</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 11:47, 17 August 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 36:</td>
<td colspan="2" class="diff-lineno">Line 36:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** 3x with 30uL BufferC+0,1%Triton (500mM imidazole)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** 3x with 30uL BufferC+0,1%Triton (500mM imidazole)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** store the supernatants for use in SDS-gel electrophoresis</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** store the supernatants for use in SDS-gel electrophoresis</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;">==Vectors==</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;">*Isolated plasmid using 'Sigma Gen Elute plasmid miniprep kit'</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;">::-performed additional wash step in both samples.</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;">::-eluted in 50ul dH2O</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>==Dry==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Dry==</div></td></tr>
</table>Franshttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_July_2009&diff=19037&oldid=prevVerhoeven1981 at 10:17, 13 July 20092009-07-13T10:17:12Z<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 10:17, 13 July 2009</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>{{Team:Groningen/Notebook/Day/Header}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Groningen/Notebook/Day/Header}}</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">Dry</del>==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==<ins class="diffchange diffchange-inline">Wet</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">A start was made with modelling arsenic uptake on the [[Team:Groningen/Modelling|Modelling]] page. It was mentioned by Martijn (iGEM team member of last year) that there exists software to give an estimate of binding affinities, in case we can't find specific ones (he hasn't been able to find the right link yet though).</del></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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Also, a planning </del>for <del class="diffchange diffchange-inline">Construction 1 was made, focussing on arsenic for now. During the meeting the lab workers agreed that apart from arsenic the next metals to look at would be Zinc and Copper.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===GVP Cluster===</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 class="diffchange diffchange-inline">'''Colonies grown </ins>for <ins class="diffchange diffchange-inline">30 hours'''</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 class="diffchange diffchange-inline">Wet=</del>=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">From the transformed E. coli TOP10 cells with [http://partsregistry.org/wiki/index.php?title</ins>=<ins class="diffchange diffchange-inline">Part:BBa_J23109 BBa_J23109], [http://partsregistry.org/wiki/index.php?title</ins>=<ins class="diffchange diffchange-inline">Part:BBa_J23100 BBa_J23100] and [http://partsregistry.org/wiki/index.php?title</ins>=<ins class="diffchange diffchange-inline">Part:BBa_J23106 BBa_J23106] three single colonies of each were chosen to plate anew on TY-agar-amp. plates. After hitting the colony, the cells were swiped in a single straight line, the tip heated and a new stripe through the old was made. This was repeated three times in total. The plates were stored at 37C over night. </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 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;">===Transporters===</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Purification of HmtA (PA Q9I147 6xHis Tag)'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Purification of HmtA (PA Q9I147 6xHis Tag)'''</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 36:</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>*** store the supernatants for use in SDS-gel electrophoresis</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** store the supernatants for use in SDS-gel electrophoresis</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">'Colonies grown for 30 hours'''</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">==Dry==</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 class="diffchange diffchange-inline">A start was made with modelling arsenic uptake on the [[Team:Groningen/Modelling|Modelling]] page. It was mentioned by Martijn (iGEM team member of last year) that there exists software to give an estimate of binding affinities, in case we can</ins>'<ins class="diffchange diffchange-inline">t find specific ones (he hasn</ins>'<ins class="diffchange diffchange-inline">t been able to find the right link yet though).</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 class="diffchange diffchange-inline">From the transformed E. coli TOP10 cells with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109]</del>, <del class="diffchange diffchange-inline">[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] three single colonies of each were chosen to plate anew </del>on <del class="diffchange diffchange-inline">TY-agar-amp</del>. <del class="diffchange diffchange-inline">plates. After hitting </del>the <del class="diffchange diffchange-inline">colony, </del>the <del class="diffchange diffchange-inline">cells were swiped in a single straight line, </del>the <del class="diffchange diffchange-inline">tip heated </del>and <del class="diffchange diffchange-inline">a new stripe through the old was made. This was repeated three times in total. The plates were stored at 37C over night</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Also</ins>, <ins class="diffchange diffchange-inline">a planning for Construction 1 was made, focussing </ins>on <ins class="diffchange diffchange-inline">arsenic for now</ins>. <ins class="diffchange diffchange-inline">During </ins>the <ins class="diffchange diffchange-inline">meeting </ins>the <ins class="diffchange diffchange-inline">lab workers agreed that apart from arsenic </ins>the <ins class="diffchange diffchange-inline">next metals to look at would be Zinc </ins>and <ins class="diffchange diffchange-inline">Copper</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;"></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>{{Team:Groningen/Notebook/Day/Footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Groningen/Notebook/Day/Footer}}</div></td></tr>
</table>Verhoeven1981http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_July_2009&diff=17203&oldid=prevJolandaWitteveen: /* Wet */2009-07-09T12:22:37Z<p><span class="autocomment">Wet</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 12:22, 9 July 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 11:</td>
<td colspan="2" class="diff-lineno">Line 11:</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>Growing cells and purification </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Growing cells and purification </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* Resuspend pellet in 50mM <del class="diffchange diffchange-inline">Kpi </del>pH7,0 with 1M NaCl</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Resuspend pellet in 50mM <ins class="diffchange diffchange-inline">KPi </ins>pH7,0 with 1M NaCl</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* Spin down for 25 min at 80 000 rpm</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Spin down for 25 min at 80 000 rpm <ins class="diffchange diffchange-inline">(TLA 100.4)</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Add 1 ml solubilazation buffer to the pellet </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Add 1 ml solubilazation buffer to the pellet </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Solubilzation buffer (for 25ml)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** Solubilzation buffer (for 25ml)</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*** 1,25 ml <del class="diffchange diffchange-inline">Kpi </del>(1M)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*** 1,25 ml <ins class="diffchange diffchange-inline">KPi </ins>(1M)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** 2 ml NaCl ( 5M)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** 2 ml NaCl ( 5M)</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*** 6,67 ml <del class="diffchange diffchange-inline">Gly </del>(75%)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*** 6,67 ml <ins class="diffchange diffchange-inline">Glycerol </ins>(75%)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** 15,08 ml water</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** 15,08 ml water</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** 30 min on ice</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** 30 min on ice</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* Spin down for 25 min at 80 000 rpm</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Spin down for 25 min at 80 000 rpm <ins class="diffchange diffchange-inline">(TLA 100.4)</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Wash the Ni-NTA beads; </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Wash the Ni-NTA beads; </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** 30 ul Ni-NTA in 1 ml buffer A,</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** 30 ul Ni-NTA in 1 ml buffer A,</div></td></tr>
</table>JolandaWitteveenhttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_July_2009&diff=17179&oldid=prevVerhoeven1981: /* Wet */2009-07-09T11:58:18Z<p><span class="autocomment">Wet</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 11:58, 9 July 2009</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;"><div>'''Colonies grown for 30 hours'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Colonies grown for 30 hours'''</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>From the transformed E. coli TOP10 cells with BBa_J23109, BBa_J23100 and BBa_J23106 three single colonies of each were chosen to plate anew on TY-agar-amp. plates. After hitting the colony, the cells were swiped in a single straight line, the tip heated and a new stripe through the old was made. This was repeated three times in total. The plates were stored at 37C over night. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>From the transformed E. coli TOP10 cells with <ins class="diffchange diffchange-inline">[http://partsregistry.org/wiki/index.php?title=Part:</ins>BBa_J23109 <ins class="diffchange diffchange-inline">BBa_J23109]</ins>, <ins class="diffchange diffchange-inline">[http://partsregistry.org/wiki/index.php?title=Part:</ins>BBa_J23100 <ins class="diffchange diffchange-inline">BBa_J23100] </ins>and <ins class="diffchange diffchange-inline">[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 </ins>BBa_J23106<ins class="diffchange diffchange-inline">] </ins>three single colonies of each were chosen to plate anew on TY-agar-amp. plates. After hitting the colony, the cells were swiped in a single straight line, the tip heated and a new stripe through the old was made. This was repeated three times in total. The plates were stored at 37C over night. </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;"></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>{{Team:Groningen/Notebook/Day/Footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Groningen/Notebook/Day/Footer}}</div></td></tr>
</table>Verhoeven1981http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_July_2009&diff=17150&oldid=prevVerhoeven1981: /* Wet */2009-07-09T11:28:56Z<p><span class="autocomment">Wet</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 11:28, 9 July 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 35:</td>
<td colspan="2" class="diff-lineno">Line 35:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** 3x with 30uL BufferC+0,1%Triton (500mM imidazole)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** 3x with 30uL BufferC+0,1%Triton (500mM imidazole)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** store the supernatants for use in SDS-gel electrophoresis</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** store the supernatants for use in SDS-gel electrophoresis</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;">'''Colonies grown for 30 hours'''</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;">From the transformed E. coli TOP10 cells with BBa_J23109, BBa_J23100 and BBa_J23106 three single colonies of each were chosen to plate anew on TY-agar-amp. plates. After hitting the colony, the cells were swiped in a single straight line, the tip heated and a new stripe through the old was made. This was repeated three times in total. The plates were stored at 37C over night. </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;"></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>{{Team:Groningen/Notebook/Day/Footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Groningen/Notebook/Day/Footer}}</div></td></tr>
</table>Verhoeven1981http://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_July_2009&diff=17101&oldid=prevJolandaWitteveen: /* Dry */2009-07-09T10:18:49Z<p><span class="autocomment">Dry</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 10:18, 9 July 2009</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 5:</td>
<td colspan="2" class="diff-lineno">Line 5:</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>Also, a planning for Construction 1 was made, focussing on arsenic for now. During the meeting the lab workers agreed that apart from arsenic the next metals to look at would be Zinc and Copper.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Also, a planning for Construction 1 was made, focussing on arsenic for now. During the meeting the lab workers agreed that apart from arsenic the next metals to look at would be Zinc and Copper.</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;">==Wet==</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;">'''Purification of HmtA (PA Q9I147 6xHis Tag)'''</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;">Growing cells and purification </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;">* Resuspend pellet in 50mM Kpi pH7,0 with 1M NaCl</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;">* Spin down for 25 min at 80 000 rpm</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;">* Add 1 ml solubilazation buffer to the pellet </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;">** Solubilzation buffer (for 25ml)</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;">*** 1,25 ml Kpi (1M)</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;">*** 2 ml NaCl ( 5M)</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;">*** 6,67 ml Gly (75%)</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;">*** 15,08 ml water</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;">*** 30 min on ice</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;">* Spin down for 25 min at 80 000 rpm</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;">* Wash the Ni-NTA beads; </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;">** 30 ul Ni-NTA in 1 ml buffer A,</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;">* Spin 2 min 3500 rpm, remove supernatant </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;">* Add supernatant of protein to epi with the Ni-NTA pellet</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;">* Incubate for 2 h, shaking</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;">* Spin 4 min at 3500 rpm with table spin</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;">* Wash</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;">** 2x with 1ml buffer B+0,1% triton (20mM imidazole)</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;">*** store the supernatants for use in SDS-gel electrophoresis</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;">** 2x with 1ml bufferB+01,% Triton (40 mM imidazole)</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;">*** store the supernatants for use in SDS-gel electrophoresis</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;">* Elute</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;">** 3x with 30uL BufferC+0,1%Triton (500mM imidazole)</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;">*** store the supernatants for use in SDS-gel electrophoresis</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;"></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>{{Team:Groningen/Notebook/Day/Footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Groningen/Notebook/Day/Footer}}</div></td></tr>
</table>JolandaWitteveenhttp://2009.igem.org/wiki/index.php?title=Team:Groningen/Notebook/8_July_2009&diff=16748&oldid=prevJaspervdg: Dry log.2009-07-08T16:52:13Z<p>Dry log.</p>
<p><b>New page</b></p><div>{{Team:Groningen/Notebook/Day/Header}}<br />
<br />
==Dry==<br />
A start was made with modelling arsenic uptake on the [[Team:Groningen/Modelling|Modelling]] page. It was mentioned by Martijn (iGEM team member of last year) that there exists software to give an estimate of binding affinities, in case we can't find specific ones (he hasn't been able to find the right link yet though).<br />
<br />
Also, a planning for Construction 1 was made, focussing on arsenic for now. During the meeting the lab workers agreed that apart from arsenic the next metals to look at would be Zinc and Copper.<br />
<br />
{{Team:Groningen/Notebook/Day/Footer}}</div>Jaspervdg