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Team:BIOTEC Dresden/Protocols:Ligation
From 2010.igem.org
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<li>10X T4 DNA ligase buffer</li> | <li>10X T4 DNA ligase buffer</li> | ||
<li>T4 DNA ligase</li> | <li>T4 DNA ligase</li> | ||
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</div> | </div> | ||
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<h2>Procedure</h2> | <h2>Procedure</h2> | ||
<ul> | <ul> | ||
| + | <li>How to calculate the Insert Amount, if, for example, a 6:1 molar ratio is decided upon</li> | ||
<li>Into a <span class="markup volume">1.5mL</span> add the following:</li> | <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><span class="markup volume">2.0µL</span> 10X T4 DNA Ligase Buffer</li> | ||
Revision as of 21:09, 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
- How to calculate the Insert Amount, if, for example, a 6:1 molar ratio is decided upon
- 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
