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Team:Hawaii/Procols/DNA ligation
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(New page: ===Protocol=== #Combine 4 ul of construct one with 4 ul of construct 2. #Add 1 ul of nanopure H<sub>2</sub>0. #Add 10 ul of 2x quick ligation reaction Buffer. In order to avoid shearing...)
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(New page: ===Protocol=== #Combine 4 ul of construct one with 4 ul of construct 2. #Add 1 ul of nanopure H<sub>2</sub>0. #Add 10 ul of 2x quick ligation reaction Buffer. In order to avoid shearing...)
Newer edit →
Revision as of 22:01, 9 July 2008
Protocol
- Combine 4 ul of construct one with 4 ul of construct 2.
- Add 1 ul of nanopure H20.
- Add 10 ul of 2x quick ligation reaction Buffer. In order to avoid shearing the DNA, mix by resuspending very slowly.
- Add 1 ul of Quick T4 DNA ligase and mix thoroughly by resuspending very slowly.
- Incubate at room temperature for 5 minutes.
- Cool on ice then transform, or store at -20oC
Notes:
- Adjust amount of DNA as necessary. For ligation of 2 pieces of DNA, use 1:1 ratio of parts. For ligation of insert to vector, use 3:1 ratio of insert to vector. ~180ng of DNA can be ligated using the above protocol.
- Reference: Quick Ligation Kit from NEB.
