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Team:BIOTEC Dresden/Protocols:Ligation
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Revision as of 21:10, 25 October 2010 by Lucas.schirmer (Talk | contribs)
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
