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2010-10-26T17:13:44Z
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<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;">Protocol 13: Vector Dephosphorylation</del></div></td><td colspan="2"> </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: #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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Reagents:</del></div></td><td colspan="2"> </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;">* Antarctic phosphatase</del></div></td><td colspan="2"> </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;">* 10x Antarctic phosphatase buffer</del></div></td><td colspan="2"> </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: #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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Procedure:</del></div></td><td colspan="2"> </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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">* Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5ug of DNA cut with any restriction endonuclease in any buffer.</del></div></td><td colspan="2"> </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;">* Add 1ul of Antarctic phosphatase and mix.</del></div></td><td colspan="2"> </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;">* Incubate 5 minutes at 37<sup>o</sup>C.</del></div></td><td colspan="2"> </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;">* Heat inactivate for 5 minutes at 65<sup>o</sup>C.</del></div></td><td colspan="2"> </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;">* Proceed with ligation.</del></div></td><td colspan="2"> </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: #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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Notes:</del></div></td><td colspan="2"> </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: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">* Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh. </del></div></td><td colspan="2"> </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;">* It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency. </del></div></td><td colspan="2"> </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;">* Try without dephosphorylation first, and minimize exposure to phosphatase if you must resort to using it. </del></div></td><td colspan="2"> </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;">** See also Sambrook.</del></div></td><td colspan="2"> </td></tr>
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<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;">[[Team:Alberta/Notebook/protocols| Back]]</del></div></td><td colspan="2"> </td></tr>
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Wiltshir
http://2010.igem.org/wiki/index.php?title=Protocol/13&diff=152637&oldid=prev
Anh at 01:37, 26 October 2010
2010-10-26T01:37:15Z
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<td colspan='2' style="background-color: white; color:black;">Revision as of 01:37, 26 October 2010</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Alberta/beginMainContent}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Alberta/beginMainContent}}</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Protocol 13: Vector <del class="diffchange diffchange-inline">dephosphorylation protocol</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Protocol 13: Vector <ins class="diffchange diffchange-inline">Dephosphorylation</ins></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">What you will need</del>:</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">Reagents</ins>:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Antarctic phosphatase</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Antarctic phosphatase</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* 10x Antarctic phosphatase buffer</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* 10x Antarctic phosphatase 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 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>Procedure:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Procedure:</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>* Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-<del class="diffchange diffchange-inline">5 ug </del>of DNA cut with any restriction endonuclease in any buffer</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-<ins class="diffchange diffchange-inline">5ug </ins>of DNA cut with any restriction endonuclease in any buffer<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>* Add <del class="diffchange diffchange-inline">1 ul </del>of Antarctic phosphatase and mix</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Add <ins class="diffchange diffchange-inline">1ul </ins>of Antarctic phosphatase and mix<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>* Incubate 5 <del class="diffchange diffchange-inline">min </del>at 37 C</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Incubate 5 <ins class="diffchange diffchange-inline">minutes </ins>at 37<ins class="diffchange diffchange-inline"><sup>o</sup></ins>C<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>* Heat inactivate for 5 minutes at 65 C</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Heat inactivate for 5 minutes at 65<ins class="diffchange diffchange-inline"><sup>o</sup></ins>C<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>* Proceed with ligation</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Proceed with ligation<ins class="diffchange diffchange-inline">.</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> </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">Notes:</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">* Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh. </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">* It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency. </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">* Try without dephosphorylation first, and minimize exposure to phosphatase if you must resort to using it. </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">** See also Sambrook.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">==Notes==</del></div></td><td colspan="2"> </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;">Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh. It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency. Try without dephosphorylation first, and minimize exposure to phosphatase if you must resort to using it. See also Sambrook.</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>[[Team:Alberta/Notebook/protocols| Back]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Team:Alberta/Notebook/protocols| Back]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Alberta/endMainContent}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Alberta/endMainContent}}</div></td></tr>
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Anh
http://2010.igem.org/wiki/index.php?title=Protocol/13&diff=152139&oldid=prev
Anh: New page: {{Team:Alberta/Head}} {{Team:Alberta/navbar|project=selected}} {{Team:Alberta/beginLeftSideBar}} {{Team:Alberta/endLeftSideBar}} {{Team:Alberta/beginRightSideBar}} {{Team:Alberta/endRig...
2010-10-26T00:42:13Z
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Protocol 13: Vector dephosphorylation protocol<br />
<br />
What you will need:<br />
* Antarctic phosphatase<br />
* 10x Antarctic phosphatase buffer<br />
<br />
Procedure:<br />
<br />
* Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5 ug of DNA cut with any restriction endonuclease in any buffer<br />
* Add 1 ul of Antarctic phosphatase and mix<br />
* Incubate 5 min at 37 C<br />
* Heat inactivate for 5 minutes at 65 C<br />
* Proceed with ligation<br />
<br />
==Notes==<br />
Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh. It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency. Try without dephosphorylation first, and minimize exposure to phosphatase if you must resort to using it. See also Sambrook.<br />
<br />
[[Team:Alberta/Notebook/protocols| Back]]<br />
{{Team:Alberta/endMainContent}}</div>
Anh