Team:Todai-Tokyo/Protocols/Sequencing

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Sequencing Protocol

We use the Big Dye Sequencing system with using the following conditions.

  1. Prepare the following solution:
    1. 1.8ul 5 ｘ B.D.3.1 buffer　
    2. 0.4ul B.D.3.1.　
    3. 6.3ul MilliQ　
    4. 1ul 0.15ug/ul DNA
    5. 0.5ul 3.2pmol/ul　primer
  2. Amplify using the following PCR program
    1. 96ºC 2min
    2. 96ºC 10sec
    3. 55ºC 5sec
    4. 60ºC 3min
    5. Repeat 2-4 29times
    6. 25ºC pause
  3. Add 0.5ul Shrimp Alkaline Phosphatase
  4. Incubate at 37ºC for 1h
  5. Add 1ul of 3M KOAc or NaOAc
  6. Mix and transfer to 1.5ml eppendorf
  7. Add 25ul of 100% EtOH　and mix well
  8. Spin for 10min at 20,000g, 4ºC
  9. Discard supernatant and dry pellet
  10. Add 15ul of HiDi Buffer
  11. Transfer to PCR tubes to sequence (use the sequencer in the lab)
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