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Team:BIOTEC Dresden/Protocols:Ligation
From 2010.igem.org
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
