Science Gateway > Protocols > Cell Biology Protocols - Table of Contents
Star Republic: Guide for Biologists

Sequencing (ABI Big Dye Terminator)

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:

Reagent Quantity
(Half-reactions)
Quantity
(Full-reactions)
Template DNA 4 ml 8 ml
Big Dye Terminator reaction mix 4 ml 8 ml
Primer (3.2 pmol/ml) 1 ml 2 ml
ddH2O 1 ml 2 ml

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

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.