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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:::''Reference: Quick Ligation Kit from NEB.''
:::''Reference: Quick Ligation Kit from NEB.''
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* 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.
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:::''Reference: OpenWetWare''

Latest revision as of 05:31, 13 August 2008

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 -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.
  • 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
Retrieved from "http://2008.igem.org/Team:Hawaii/Procols/DNA_ligation"