Seaquencing

Preparation

• ・B.D.3.1.
• ・5xB.D.3.1. buffer
• ・PHOSPHATASE ALKALINE shrimp
• ・3M KOAc or NaOAc
• ・100% EtOH
• ・HiDi

Procedure

• 1. Prepare leading solution in 1.5 ml eppendorf-tube(eppen) below
  • 5xB.D.3.1. buffer 1.8 ul x (sumple amount + 1)
  • B.D.3.1. 0.4 ul x (-)
  • MilliQ 6.3 ul x (-)
• 2. Put solutions below in 8-series-tube
  • 1 ul deluted solution of plasmid (0.15 ug/ul)
  • 0.5 ul 5′ primer solution or 3′ primer solution (3.2 pmol/ul)
  • 8.5 ul leading solution
• 3. PCR by BIGDYE program
  • 1. 96 degrees C for 2 minutes
  • 2. 96 degrees C for 10 seconds
  • 3. 55 degrees C for 5 seconds
  • 4. 60 degrees C for 3 minutes
  • 5. repeat 2,3,4 29 times
  • 6. 25 degrees C forever
• 4. Add 0.5 ul PHOSPHATASE ALKALINE shimp
• 5. Incubate at 37 degrees C for 1 hour
• 6. Add 1 ul 3M KOAcor NaOAc
• 7. Tapping
• 8. Remove 1.5 ml to the other tube and add 25 ul 100% EtOH
• 9. Tapping or boltex
• 10. Centrifuge (20000xg, 4 degrees C, 10 minutes)
• 11. Remove supernatant
• 12. Air dry
• 13. Add 15 ul HiDi, tap well and dissolve all pellets
• 14. Remove to 8-series-tube. (In order to do 16 sequencings all together, put HiDi in the tube with no sumple)
• 15. Put into Sequencing device. (You can store the sample at -20 degrees C and read sequencing results next day.)