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Team:BIOTEC Dresden/Protocols:Ligation
From 2010.igem.org
(Difference between revisions)
(New page: {{Biotec_Dresden/Header}} <html> <body> <div id="content_prim"> <h3>Hint</h3> <p>Try different molar ratios of vector to plasmid to increase ligation effficiency</p> <p>The sticky end...) |
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<li>How to calculate the Insert Amount, if, for example, a 6:1 molar ratio is decided upon</li> | <li>How to calculate the Insert Amount, if, for example, a 6:1 molar ratio is decided upon</li> | ||
| + | </ul> | ||
| + | </div> | ||
| + | |||
| + | <div class="visualClear"></div> | ||
| + | <div id="procedure"> | ||
| + | <h2>Procedure</h2> | ||
| + | <ul> | ||
| + | <li>Into a <span class="markup volume">1.5mL</span> add the following:</li> | ||
| + | <li><span class="markup volume">2.0µL</span> 10X T4 DNA Ligase Buffer</li> | ||
| + | <li>volume of insert that corresponds to the determined molar ratio</li> | ||
| + | <li>volume of vector that corresponds to the determined molar ratio</li> | ||
| + | <li><span class="markup volume">0.5µL</span> T4 DNA Ligase</li> | ||
| + | <li>Adjust final volume to <span class="markup volume">20µL</span> with ultra pure water</li> | ||
| + | <li>Vortex and pulse spin down</li> | ||
| + | <li>Incubate at <span class="markup temp">room temperature</span> for <span class="markup time">1 hour</span></li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
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</body> | </body> | ||
</html> | </html> | ||
| - | + | [[Category:BIOTEC Dresden/Protocol]] | |
{{Biotec_Dresden/Bottom}} | {{Biotec_Dresden/Bottom}} | ||
Revision as of 21:08, 25 October 2010
Hint
Try different molar ratios of vector to plasmid to increase ligation effficiency
The sticky ends of both the digested insert and vector DNA must be comptabile
Materials
- ultra-pure water
- purified digested insert DNA
- purified digested vector DNA
- 10X T4 DNA ligase buffer
- T4 DNA ligase
Procedure
- Into a 1.5mL add the following:
- 2.0µL 10X T4 DNA Ligase Buffer
- volume of insert that corresponds to the determined molar ratio
- volume of vector that corresponds to the determined molar ratio
- 0.5µL T4 DNA Ligase
- Adjust final volume to 20µL with ultra pure water
- Vortex and pulse spin down
- Incubate at room temperature for 1 hour
