Knight:Annealing and cloning oligos

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in progress, this protocol hasn't worked yet; use at your own risk!!!


This is a protocol to anneal and ligate oligo's to construct short DNA fragments for cloning (~100 bp)


  • 3 oligo pairs with 4-5 base pair overlaps
    • Austin suggests 6 bp overlaps.
  • T4 DNA ligase
  • T4 DNA ligase buffer
  • T4 polynucleotide kinase


  1. Resuspend primers.
  2. Kinase treat each oligo using the following reaction mix for each primer.
    • 2 μL primer (5 μM final concentration)
    • 1 μL 10X T4 DNA ligase buffer
    • 6.5 μL H2O
    • 0.5 μL T4 polynucleotide kinase
  3. Incubate at 37 °C for 1 hr 30 mins
  4. Heat kill at 65 °C for 20 mins
  5. Mix all 10μL of kinase treated oligo with its pair to make duplex oligos.
  6. Heat to 70-75 °C and cool to anneal oligo pairs.
  7. Mix all 20 μL of each duplex together in one tube.
  8. Make up ligation reaction
    • 1 μL of the oligo mix
    • 50 ng digested vector backbone
      • The assembled oligo construct should have complementary ends to the prepared vector
    • 1 μL 10X T4 DNA ligase buffer
    • H2O to 10 uL
  9. Add 0.5 μL T4 DNA ligase
  10. Incubate for 1hr 30 mins at 16°C.
  11. Transform.


  • I did not include 5' phosphates on the flanking primers to decrease the likelihood of forming multimers.
  • Pete Carr recommends PCR construction and assembly since ligation methods can be unreliable.
  • Too much ligase may adversely affect the reaction.