Seq480

Cycle Sequencing with the DNA Cycler 480 (from Chris Cohick)

Add the following to a 0.5 mL thin walled PCR tube for each reaction:
Mix well, may spin briefly. Overlay the reaction mixture with 40 µL of mineral oil.
Place the tubes in the thermocycler and begin cycling with the following parameters:
1. Rapid thermal ramp to 96 ºC
2. 96 ºC for 30 seconds
3. Rapid thermal ramp to 50 ºC
4. 50 ºC for 15 seconds
5. Rapid thermal ramp to 60 ºC
6. 60 ºC for 4 minutes

Reagent	Quantity
Terminator Ready Reaction Mix	4-8 µL
Template (Template Quality and accurate Quantitation of your template is critical for this procedure)
ssDNA	50 - 100 ng
dsDNA	200-500 ng
PCR Product	30-90 ng
Primer	1.6 pmol
Deionized Water	q.s.
Total Volume	20 µL

Repeat for 25 cycles ~2.5 hours.
Rapid thermal ramp to 4 ºC and hold until ready to purify.
Spin down contents in a microcentrifuge.
For each sequencing reaction prepare a 1.5 mL microfuge tube containing 2 µL of 3 M sodium acetate pH 4.6 and 50 µL of 95% EtOH.
Pipet the contents of each reaction into one of these tubes, avoiding the mineral oil..
Mix well and place on ice for 10 minutes to precipitate the extension products.
Spin the tubes 15-30 minutes in a microfuge at maximum speed.
Carefully aspirate the supernatant with a pipette and discard. (Note: You will not be able to see a pellet. Something is wrong if you do see a pellet, most likely quantitation of your template.)
Rinse pellet with 250 µL of 70% EtOH. (Note: Elimination of salt is important for sequence quality. Do not skip this step.)
Spin for five minutes in a microfuge at maximum speed. Carefully aspirate the supernatant and discard.
Dry the pellet. Do not overdry (best: place open tube in 95°C heatblock for 1 min.)
Resuspend in 12µl Template Suppression Reagent.
Heat again for 1 min. in 95°C heatblock. Transfer into 0.5ml sequencer sample tubes. Make sure that the rubber septa can open properly (squeeze to check).