Team:UT-Tokyo/Seaquencing
From 2010.igem.org
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.)
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