Seq2400

Cycle Sequencing with the GeneAmp 2400 (from Chris Cohick)

Add the following to a 0.2 mL thin walled PCR tube for each reaction. Mix well, may spin briefly.
Place the tubes in the thermocycler and begin cycling with the following parameters:
1. Rapid thermal ramp to 96 ºC
2. 96 ºC for 10 seconds
3. Rapid thermal ramp to 50 ºC
4. 50 ºC for 5 seconds
5. Rapid thermal ramp to 60 ºC
6. 60 ºC for 4 minutes
Repeat for 25 cycles (~2.5h).

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

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.
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).