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Team:Alberta/Notebook/protocols/sequencing
Fluorescent Sequencing Reaction
Procedure:
- In a 0.2ml PCR tube, add:
| Template | 5.0ul (200 ng/ul) |
| VF primer | 1.0ul |
| Dilute buffer | 2.5ul |
| BD sequence mix | 1.5ul |
- Mix well.
- Select program 'seq-dye' on PTC 200 thermal cycler.
- Run program. It will take about 2 hours.
- Remove tube from PCR machine and transfer 10ul rxn mix to 1.5ml Eppendorf tube. Add:
| Blue NaOAc/EDTA | 1.5ul |
| 95% ethanol | 40ul |
- Let sit on ice 15 minutes.
- Centrifuge 10 minutes at max speed.
- Should see a small blue dot at bottom of tube.
- Discard supernatant.
- Wash pellet with 500ul of 70% ethanol.
- Discard supernatant and spin briefly to bring down the residual liquid. Draw liquid off with a P10 pipette tip.
- Do not disturb the pellet.
- Air dry for 10 minutes.
- Place in -20oC freezer to be delivered to MBSU 4th floor microbiology M534. Reactions delivered before 2pm will normally be returned the next day.
