MPL Protocol for DNA Sequencing using ABI Big Dye Terminator Cycle Sequencing Kit

Step 1 - Preparing the sequencing reactions for dsDNA PCR products, assuming "half-reactions".

For each reaction (template/primer combination), add the following reagents to a separate 0.2 ml microamp tube:



Terminator Ready Reaction Mix

4.0 ml


up to 4 ml (30-90 ng*)

primer (at 3.2 mM)

1.0 ml



total volume

10 ml

mix well and spin briefly


* Depends on size of PCR product; the DBS sequencing team at UCDavis suggests approximately 4 ml of a sample of dsDNA template at a concentration of 2 ng/ml per 100 bp length required for each reaction. This works well for most small to moderate size PCR products and plasmids clones. For example, for a 1.0 kb template, 4 ml of a sample with a concentration of 20 ng/ml (total of ca. 80 ng) would be required. We however, use about half of this amount for most PCR product reactions.

Step 2 - Sequencing on the PE 2400 or 9600 thermal cyclers.

Place the tubes in the thermal cycler and begin temperature cycling protocol. Program the thermocycler as as follows: 25 cycles of [96 C for 10 sec, 50 C for 5-10 sec, 60 C for 4 min], then ramp to 4 C, purify extension products as below.

Step 3 - Purifying sequencing extension products by isopropanol precipitation.

Briefly spin tubes and transfer by pipeting entire sequencing reactions into 1.5 ml microcentrifuge tubes. Then . . .

a. add 40 ml of 75% isopropanol, or 10 ml of deionized water and 30 ml of 100% isopropanol.

b. mix by vortexing briefly, leave at room temperature for >15 min (and < 24 hrs) to precipitate products.

c. spin tubes for a minimum of 20 min at maximum speed in a microcentrifuge.

d. aspirate the supernatants completely with a separate pipet tip for each sample, being careful not to disturb the DNA pellet, and discard.

e. add 125 to 250 ml of 75% isopropanol to the tubes and vortex briefly, centrifuge as before for 5 min at maximum speed, and aspirate the supernatants as in step d.

f. dry the samples in a vacuum centrifuge for 10 - 15 minutes (to dryness), or place on a thermal cycler heating block for 1 min at 95 C, and store at -20 C until ready for electrophoresis.

Step 4 - Preparation for electrophoresis.

Redissolve each sample pellet in 3 ml loading buffer [deionized formamide/25 mM EDTA (pH 8.0) with blue dextran (50 mg/ml), at 5:1 vol/vol] immediately before use. Vortex and spin samples. Heat samples at 95 C for 2 min, then immediately place on ice until ready to load. Load 1 - 2 ml per sample.

(© J. A. Wheeler and M. F. Wojciechowski, Molecular Phylogenetics Lab, UC Berkeley, 1998)