Difference between revisions of "Team:Freiburg/Protocols/Ligation"

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<h1 class="sectionedit1"><a name="ligation" id="ligation">Ligation</a></h1>
 
<h1 class="sectionedit1"><a name="ligation" id="ligation">Ligation</a></h1>
 
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Classical cloning as well as Gibson Assembly require the action of ligases to covalently assemble DNA fragments. Before ligation, those fragments anneal to each other due to complementary overhangs.
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<th class="col0 leftalign"> amount </th><th class="col1 leftalign"> Substance              </th>
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<th> Amount </th><th class="col1 leftalign"> Ingredient </th>
 
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<td class="col0 leftalign"> 50 ng  </td><td class="col1 leftalign"> Backbone              </td>
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<td> 50 ng  </td><td class="col1 leftalign"> Backbone              </td>
 
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<td class="col0"> 3fold molar amount</td><td class="col1 leftalign"> Insert                </td>
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<td> 3 fold molar amount</td><td class="col1 leftalign"> Insert                </td>
 
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<td class="col0 leftalign"> 2 µl  </td><td class="col1 leftalign"> 10X T4-Ligase Buffer  </td>
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<td> 2 µl  </td><td class="col1 leftalign"> 10X T4-Ligase Buffer  </td>
 
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<td class="col0 leftalign"> 1 µl  </td><td class="col1 leftalign"> T4-Ligase              </td>
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<td> 1 µl  </td><td class="col1 leftalign"> T4-Ligase              </td>
 
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<td class="col0 leftalign"> up to 20 µl  </td><td class="col1 leftalign"> H2O                   </td>
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<td> ad 20 µl  </td><td class="col1 leftalign"> dH<sub>2</sub>O                   </td>
 
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30 min at RT  
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Incubate for 30 min at RT  
 
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<h5><a name="equation_for_insert_calculation" id="equation_for_insert_calculation">Equation for insert calculation</a></h5>
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<h3>Calculating the amount of insert</h3>
 
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<a href="/igem2015/lib/exe/fetch.php?media=files:protocols:ligation.xlsx" class="media mediafile mf_xlsx" title="files:protocols:ligation.xlsx">Ligation</a>
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<a href="/igem2015/lib/exe/fetch.php?media=files:protocols:ligation.xlsx" class="media mediafile mf_xlsx" title="files:protocols:ligation.xlsx">Ligation Excel Sheet</a>
 
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Latest revision as of 07:06, 20 November 2015

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Ligation

Classical cloning as well as Gibson Assembly require the action of ligases to covalently assemble DNA fragments. Before ligation, those fragments anneal to each other due to complementary overhangs.

Amount Ingredient
50 ng Backbone
3 fold molar amount Insert
2 µl 10X T4-Ligase Buffer
1 µl T4-Ligase
ad 20 µl dH2O

Incubate for 30 min at RT

Calculating the amount of insert

Ligation Excel Sheet

required Volume of Insert = 3 x Volume(Bb) x length(In) x concentration(Bb) / [ length(Bb) x concentration(In) ]

In short: V(In) = 3 x V(Bb) x l(In) x c(Bb) / [ l(Bb) x c(In) ]