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1. Quantitate purified DNA template. For PCR products less than 1 kb,
use 10-20 ng template for every 100 bp. For PCR products over 1 kb and
double stranded DNA, use 200-500 ng DNA. Adjust concentration and use
4 ml for "half-reactions" and 8
ml for "full-reactions".
2. Setup in 0.2 ml PCR tubes or 96 well PCR plate:
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Reagent
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Quantity
(Half-reactions)
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Quantity
(Full-reactions)
|
|
Template DNA
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4 ml
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8 ml
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Big Dye Terminator reaction mix
|
4 ml
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8 ml
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Primer (3.2 pmol/ml)
|
1 ml
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2 ml
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ddH2O
|
1 ml
|
2 ml
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3. Program PCR using the following cycling conditions:
1. 94 °C 5 minutes
2. 96 °C 10 seconds
3. 50 °C 4 seconds
4. 60 °C 4 minutes
5. Repeat 2-4 25-30 cycles
6. hold at 4 °C
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4. Unincorporated Big Dye need to be removed for sequencing gel.
Purifying sequencing products by isopropanol precipitation:
5. For each "half-reaction", add 40 ml of
75% isopropanol.
6. For reactions in a PCR tube, mix by vortexing briefly,
leave at room temperature for > 15 minutes (and < 24 hours) to precipitate
products.
7. Centrifuge at maximum speed for 20 minutes in a microfuge.
8. Remove carefully the supernatants with a separate pipet tip for each sample.
9. Add 150 ml of 75% isopropanol to the tubes
and vortex briefly. Centrifuge at maximum speed for 5 minutes. Discard supernatants.
10. Allow the samples to air dry or dry in a vacuum centrifuge.
6a. For reactions in 96-well PCR plate, seal the plate with aluminum tape and make
sure each well are well sealed. Invert tray a few times to mix. Leave at room temperature for > 15 minutes (and < 24 hours) to precipitate
products.
7a. Centrifuge at 2000 g for 45 minutes.
8a. Discard supernatant by inverting plate over sink.
9a. Centrifuge at 700 g for 1 minute. Discard supernatant by inverting plate over sink.
10a. Allow the samples to air dry.
11. Resuspend sequencing products in 3 ml
of loading buffer (5 volume formamide : 1 volume 25 mM EDTA (pH 8.0) with
50 mg/ml blue dextran) for "half-reactions".
12. Incubate on PCR machine 90 °C for 2 minutes to denature. Place on ice
until loaded onto gel.
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