Nmbhat:3-way ligation: Difference between revisions
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#Vortex each item before adding it to ensure that it is uniformly mixed. | #Vortex each item before adding it to ensure that it is uniformly mixed. | ||
#Add them to the tube in the order given above. When pipetting the ligase, be sure not to get too much glycerol on the sides of the pipet tip. | #Add them to the tube in the order given above. When pipetting the ligase, be sure not to get too much glycerol on the sides of the pipet tip. | ||
#Let sit at 22.5°C (room temperature) for | #Let sit at 22.5°C (room temperature) for 30 min. | ||
#Place the tube at 65°C to denature the ligase. | #Place the tube at 65°C for 10 min to denature the ligase. | ||
#Store at -20°C | #Store at -20°C | ||
Latest revision as of 12:51, 18 June 2007
Materials
- T4 DNA Ligase
- 10x T4 DNA Ligase Buffer
- Deionized, sterile H2O
- Purified, linearized vector (likely in H2O or EB)
- Purified, linearized insert "A" (ditto)
- Purified, linearized insert "B" (ditto)
Procedure
10.5μL ligation mix
- 1.0 µL T4 ligase buffer
- 3.0 µL insert A
- 3.0 µL insert B
- 3.0 µL vector
- 0.5 µL T4 DNA ligase
Method
- Vortex each item before adding it to ensure that it is uniformly mixed.
- Add them to the tube in the order given above. When pipetting the ligase, be sure not to get too much glycerol on the sides of the pipet tip.
- Let sit at 22.5°C (room temperature) for 30 min.
- Place the tube at 65°C for 10 min to denature the ligase.
- Store at -20°C
