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Team:TU Darmstadt/Notebook/Methods/DNA ligation
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| - | <!--TYPO3SEARCH_begin--><div id="c86" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">DNA | + | <!--TYPO3SEARCH_begin--><div id="c86" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">DNA Ligation</h1></div><div><p><b>Short explanation:</b></p></div><div><p>DNA ligation is necessary to assemble digested DNA parts into a vector. The cut ends generated by restriciton enzymes are put together by DNA ligase.</p></div><div></div><div><p><b>Procedure: </b>20 µL batch</p></div><div><p>- T4 Ligase Buffer 2 µL |
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<p>- T4 Ligase 1 µL</p></div><div><p>- digested vectors 25 ng (Molar ratio = Insert : backbone 3:1)</p></div><div><p>- add ddH2O to 20µL</p></div><div><p>- Incubate at 16°C overnight, alternatively incubate at room temperature for 30 min (results might be worse)</p></div><div><p>- Inactivate 20min at 80°C</p></div></div><!--TYPO3SEARCH_end--> | <p>- T4 Ligase 1 µL</p></div><div><p>- digested vectors 25 ng (Molar ratio = Insert : backbone 3:1)</p></div><div><p>- add ddH2O to 20µL</p></div><div><p>- Incubate at 16°C overnight, alternatively incubate at room temperature for 30 min (results might be worse)</p></div><div><p>- Inactivate 20min at 80°C</p></div></div><!--TYPO3SEARCH_end--> | ||
Latest revision as of 00:31, 18 October 2014
DNA Ligation
Short explanation:
DNA ligation is necessary to assemble digested DNA parts into a vector. The cut ends generated by restriciton enzymes are put together by DNA ligase.
Procedure: 20 µL batch
- T4 Ligase Buffer 2 µL
- T4 Ligase 1 µL
- digested vectors 25 ng (Molar ratio = Insert : backbone 3:1)
- add ddH2O to 20µL
- Incubate at 16°C overnight, alternatively incubate at room temperature for 30 min (results might be worse)
- Inactivate 20min at 80°C
