Team:Todai-Tokyo/Protocols/Sequencing

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


 * 1) Prepare the following solution:
 * 2) 1.8ul 5 ｘ B.D.3.1 buffer
 * 3) 0.4ul B.D.3.1.
 * 4) 6.3ul MilliQ
 * 5) 1ul 0.15ug/ul DNA
 * 6) 0.5ul 3.2pmol/ul　primer
 * 7) Amplify using the following PCR program
 * 8) 96ºC 2min
 * 9) 96ºC 10sec
 * 10) 55ºC 5sec
 * 11) 60ºC 3min
 * 12) Repeat 2-4 29times
 * 13) 25ºC pause
 * 14) Add 0.5ul Shrimp Alkaline Phosphatase
 * 15) Incubate at 37ºC for 1h
 * 16) Add 1ul of 3M KOAc or NaOAc
 * 17) Mix and transfer to 1.5ml eppendorf
 * 18) Add 25ul of 100% EtOH　and mix well
 * 19) Spin for 10min at 20,000g, 4ºC
 * 20) Discard supernatant and dry pellet
 * 21) Add 15ul of HiDi Buffer
 * 22) Transfer to PCR tubes to sequence (use the sequencer in the lab)