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Team:Heidelberg/Project/Capsid Shuffling/ViroBytes - Revision history
2024-03-28T11:19:53Z
Revision history for this page on the wiki
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http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=208395&oldid=prev
Rudolf: /* Protocols */
2010-10-28T03:57:05Z
<p><span class="autocomment">Protocols</span></p>
<table style="background-color: white; color:black;">
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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 03:57, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 141:</td>
<td colspan="2" class="diff-lineno">Line 141:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*heat on the block to 95degrees for 2 minutes and let cool slowly to room temperature</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*heat on the block to 95degrees for 2 minutes and let cool slowly to room temperature</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>'''Anchor docking'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===</ins>'''Anchor docking'''<ins class="diffchange diffchange-inline">===</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>*after washing the beads and aspiration of the cleared solution the beads are ready for adding of the Anchor </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*after washing the beads and aspiration of the cleared solution the beads are ready for adding of the Anchor </div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 151:</td>
<td colspan="2" class="diff-lineno">Line 151:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*applying magnet, wash twice with 80ul of wash/binding buffer (0.5 M NaCl; 20 mM Tris HCl (pH 7.5); 1 mM EDTA) and once with 80ul of 1X T4 ligase buffer </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*applying magnet, wash twice with 80ul of wash/binding buffer (0.5 M NaCl; 20 mM Tris HCl (pH 7.5); 1 mM EDTA) and once with 80ul of 1X T4 ligase buffer </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>'''BioByte-like assembly'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===</ins>'''BioByte-like assembly'''<ins class="diffchange diffchange-inline">===</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>*Bead preparation </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Bead preparation </div></td></tr>
</table>
Rudolf
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=208170&oldid=prev
Rudolf: /* ViroByte construction */
2010-10-28T03:52:17Z
<p><span class="autocomment">ViroByte construction</span></p>
<table style="background-color: white; color:black;">
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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 03:52, 28 October 2010</td>
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<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>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*digestion with Bsa1</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*digestion with Bsa1 <ins class="diffchange diffchange-inline">- leave digesting longer than in standard protocol (at least ~3hrs)</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>==='''Anchor preparation'''===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==='''Anchor preparation'''===</div></td></tr>
</table>
Rudolf
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=208063&oldid=prev
Rudolf: /* ViroByte construction */
2010-10-28T03:48:40Z
<p><span class="autocomment">ViroByte construction</span></p>
<table style="background-color: white; color:black;">
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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 03:48, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 126:</td>
<td colspan="2" class="diff-lineno">Line 126:</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>*perform ViroByte Phusion HiFi Hot Start PCR to obtain desired fragments with complementary ends</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*perform ViroByte Phusion HiFi Hot Start PCR to obtain desired fragments with complementary ends</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;"><div>**98°C 30s 1x</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**98°C 30s 1x</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**98°C 10s</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**98°C 10s</div></td></tr>
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<td colspan="2" class="diff-lineno">Line 133:</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>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*digestion with Bsa1 </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*digestion with Bsa1</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>==='''Anchor preparation'''===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==='''Anchor preparation'''===</div></td></tr>
</table>
Rudolf
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=208038&oldid=prev
Rudolf: /* Protocols */
2010-10-28T03:47:44Z
<p><span class="autocomment">Protocols</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
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<col class='diff-marker' />
<col class='diff-content' />
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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 03:47, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 123:</td>
<td colspan="2" class="diff-lineno">Line 123:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocols==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocols==</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>'''ViroByte construction'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===</ins>'''ViroByte construction'''<ins class="diffchange diffchange-inline">===</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>*perform ViroByte PCR to obtain desired fragments with complementary ends </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*perform ViroByte <ins class="diffchange diffchange-inline">Phusion HiFi Hot Start </ins>PCR to obtain desired fragments with complementary ends</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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">**98°C 30s 1x</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">**98°C 10s</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">**72°C 45s 35x</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">**72°C 10min 1x</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">**4°C hold </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"> </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>*digestion with Bsa1 </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*digestion with Bsa1 </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>'''Anchor preparation'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===</ins>'''Anchor preparation'''<ins class="diffchange diffchange-inline">===</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>*Mix 10ul of Anchor 5 (100uM stock) with 50ul of Anchor 12689 (100ul stock); add 1.08ml of nuclease free water; mix gently</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Mix 10ul of Anchor 5 (100uM stock) with 50ul of Anchor 12689 (100ul stock); add 1.08ml of nuclease free water; mix gently</div></td></tr>
</table>
Rudolf
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=207834&oldid=prev
Rudolf: /* Protocols */
2010-10-28T03:38:34Z
<p><span class="autocomment">Protocols</span></p>
<table style="background-color: white; color:black;">
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<col class='diff-content' />
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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 03:38, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 123:</td>
<td colspan="2" class="diff-lineno">Line 123:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocols==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocols==</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">**</del>'''ViroByte construction'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''ViroByte construction'''</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>*perform ViroByte PCR to obtain desired fragments with complementary ends </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*perform ViroByte PCR to obtain desired fragments with complementary ends </div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 129:</td>
<td colspan="2" class="diff-lineno">Line 129:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*digestion with Bsa1 </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*digestion with Bsa1 </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>**'''Anchor'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">'''Anchor preparation'''</ins></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*<ins class="diffchange diffchange-inline">Mix 10ul of Anchor 5 (100uM stock) with 50ul of Anchor 12689 (100ul stock); add 1.08ml of nuclease free water; mix gently</ins></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*<ins class="diffchange diffchange-inline">heat on the block to 95degrees for 2 minutes and let cool slowly to room temperature</ins></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Anchor <ins class="diffchange diffchange-inline">docking</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>*after washing the beads and aspiration of the cleared solution the beads are ready for adding of the Anchor </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*after washing the beads and aspiration of the cleared solution the beads are ready for adding of the Anchor </div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 139:</td>
<td colspan="2" class="diff-lineno">Line 145:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*applying magnet, wash twice with 80ul of wash/binding buffer (0.5 M NaCl; 20 mM Tris HCl (pH 7.5); 1 mM EDTA) and once with 80ul of 1X T4 ligase buffer </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*applying magnet, wash twice with 80ul of wash/binding buffer (0.5 M NaCl; 20 mM Tris HCl (pH 7.5); 1 mM EDTA) and once with 80ul of 1X T4 ligase buffer </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><del class="diffchange diffchange-inline">**</del>'''BioByte-like assembly'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''BioByte-like assembly'''</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>*Bead preparation </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Bead preparation </div></td></tr>
</table>
Rudolf
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=206640&oldid=prev
Dima: /* Protocols */
2010-10-28T03:08:20Z
<p><span class="autocomment">Protocols</span></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
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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 03:08, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 123:</td>
<td colspan="2" class="diff-lineno">Line 123:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocols==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocols==</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;">**'''ViroByte construction'''</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">Anchor preparation</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">*perform ViroByte PCR to obtain desired fragments with complementary ends </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;">*digestion with Bsa1 </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;">**'''Anchor'''</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;">*after washing the beads and aspiration of the cleared solution the beads are ready for adding of the Anchor </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;">*add 40ul of the Anchor solution (~200ng/ul) </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;">*resuspend the beads and let incubate at room temperature for 15-20 minutes with occasional flicking in order to maintain the homogeneity of the solution </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;">*applying magnet, wash twice with 80ul of wash/binding buffer (0.5 M NaCl; 20 mM Tris HCl (pH 7.5); 1 mM EDTA) and once with 80ul of 1X T4 ligase buffer </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;">**'''BioByte-like assembly'''</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;">*Bead preparation </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;">*Quickly vortex beads stock solution to obtain homogeneous suspension </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;">*Aliquot 40 µl of the beads into 1.5ml Eppendorf tubes - gently tap the tubes to collect all the beads at the bottom.</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;">*Place the tubes on the magnetic rack (or place a magnet on the side of each tube) and wait for the beads to be cleared out from the solution </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;">*Carefully discard the supernatant whilst beads are still attached to the side wall </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;">*Resuspend beads in 80ul of wash/binding buffer by gentle flicking (vortexing at high speed is not recommended) </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;">*Wash the beads once with 80 µl of 1X T4 Ligase buffer, before addition of ligation premix.</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;">*Add the DNA fragments using the ligation premix: 1) 25 µl of Quick Ligase (2X buffer); 2) Amount of DNA corresponding to 200 ng. The optimal DNA concentration for the ViroByte fragments assembly was experimentally established and was in range of 200 ng per fragment. Using higher and lower DNA concentrations did not give any positive assembly results; 3) Fill up with water to obtain the 50 µl total volume.</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;">*For the ligation procedure the Quick Ligase was used that showed to be more efficient during the experimental procedure in comparison to the ordinary T4 Ligase. Optimal ligation time for the Virobytes ligation was between 15-25 minutes.</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;">*After the last DNA fragment ligation wash the beads once with Wash/Binding buffer. Moreover the DNA should be cleaved from the beads by using HindIII restriction enzyme (or the other one depending on your designed cleavage site). For the restriction a standard protocol for the DNA digestion should be used.</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>==Notebook==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Notebook==</div></td></tr>
</table>
Dima
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=206057&oldid=prev
Dima: /* Introduction */
2010-10-28T02:49:46Z
<p><span class="autocomment">Introduction</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 02:49, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 13:</td>
<td colspan="2" class="diff-lineno">Line 13:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br /></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>[[Image:<del class="diffchange diffchange-inline">Virobytes_shuffling_222</del>.jpg|thumb|350px|center|ViroBytes Assembly Scheme]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:<ins class="diffchange diffchange-inline">Virobytes_final_1</ins>.jpg|thumb|350px|center|ViroBytes Assembly Scheme]]</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>==Important facts==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Important facts==</div></td></tr>
</table>
Dima
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=205257&oldid=prev
Dima: /* Introduction */
2010-10-28T02:18:29Z
<p><span class="autocomment">Introduction</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 02:18, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 13:</td>
<td colspan="2" class="diff-lineno">Line 13:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br /></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>[[Image:<del class="diffchange diffchange-inline">Virobytes_final</del>.jpg|thumb|350px|center|ViroBytes Assembly Scheme]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:<ins class="diffchange diffchange-inline">Virobytes_shuffling_222</ins>.jpg|thumb|350px|center|ViroBytes Assembly Scheme]]</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>==Important facts==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Important facts==</div></td></tr>
</table>
Dima
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=205083&oldid=prev
Rudolf: /* ViroBytes */
2010-10-28T02:13:31Z
<p><span class="autocomment">ViroBytes</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 02:13, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 126:</td>
<td colspan="2" class="diff-lineno">Line 126:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Anchor preparation</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Anchor preparation</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;"></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;">==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;"></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;">For further reference please find our lab notebook [https://2010.igem.org/Team:Heidelberg/Notebook/Capsid_Shuffling/ViroBytes here].</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==References==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==References==</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>
Rudolf
http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Project/Capsid_Shuffling/ViroBytes&diff=204845&oldid=prev
Rudolf: /* Primers */
2010-10-28T02:08:00Z
<p><span class="autocomment">Primers</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 02:08, 28 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 106:</td>
<td colspan="2" class="diff-lineno">Line 106:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Primers===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Primers===</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">*</del>Fragment X forward [https://2010.igem.org/Team:Heidelberg/Notebook/Material/Primer#ViroBytes_primers primers]:</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Fragment X forward [https://2010.igem.org/Team:Heidelberg/Notebook/Material/Primer#ViroBytes_primers primers]:</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>5'-gctac'''ggtctc'''aNNNN+~20-25nt Cap specific</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>5'-gctac'''ggtctc'''aNNNN+~20-25nt Cap specific</div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 115:</td>
<td colspan="2" class="diff-lineno">Line 115:</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: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">*</del>Fragment X reverse primers:</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Fragment X reverse primers:</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>5'-gctac'''ggtctc'''gNNNN</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>5'-gctac'''ggtctc'''gNNNN</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;">* '''bold''' is BsaI recognition sequence</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>==Protocols==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Protocols==</div></td></tr>
</table>
Rudolf