13 Sequencing
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2. In a 0.6 ml microcentrifuge tube, combine:
- 1 )ll template solution
- 1 )ll primer solution
- 9.5 )ll reaction mix
- 8.5 )ll water
3. Overlay with a drop of mineral oil.
4. Preheat the thermal cycler to 96°C.
5. Insert the tubes and start immediately:
- 96°C for 30 sec (denaturation of template and extension products)
- rapid thermal ramp to 50°C
- 50°C for 15 sec (annealing of primer and template)
- rapid thermal ramp to 60°C
- 60°C for 4 min (extension, thereby termination and labeling)
Repeat 25 times (25 cycles).
Finally, rapid thermal ramp to 4°C.
Note: You may have to adjust plateau and ramping times depending on the
thermal cycler used. For more information, refer to the manual of your machine.
Standard reaction
6. Add 80 )ll of water to the reaction mixture (giving a total volume of 100 DNA extraction
)ll).
7. Pipet the underlying aqueous layer (containingthe extension products)
into a fresh 1.7 ml Eppendorf tube.
8. Add 100 )ll P/CIA, vortex briefly, spin using an Eppendorf centrifuge to
separate the phases, transfer the overlying aqueous layer into a new
tube.
9. Repeat the last step once.
10. Precipitate the DNA by adding 15 ).!12M sodium acetate and 300 )ll of
100% ethanol.
11. Spin for 15 min, wash the pellet with 70% ethanol and dry.
12. Redissolve the pellet in 4 )ll of the loading solution.
13. Heat the sample to 90°C for 2 min, transfer immediately on to ice.
14. Load the sample on to the automated sequencer according to manufacturer's instructions.