Team:Hawaii/Procols/DNA ligation

From 2008.igem.org

Protocol

1. Combine 4 ul of construct one with 4 ul of construct 2.
2. Add 1 ul of nanopure H₂0.
3. Add 10 ul of 2x quick ligation reaction Buffer. In order to avoid shearing the DNA, mix by resuspending very slowly.
4. Add 1 ul of Quick T4 DNA ligase and mix thoroughly by resuspending very slowly.
5. Incubate at room temperature for 5 minutes.
6. Cool on ice then transform, or store at -20^oC

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.

• The Endy lab recommends using ~10 ng vector in a reaction of with 6:1 ratio of insert to vector. Total DNA concentration should not exceed 100ng for maximum ligation efficiency.

Reference: OpenWetWare

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