Sequencing

Sequencing

Procedure Page

Sequencing is much like PCR. The differences are that instead of creating double-stranded copies, we create single-stranded copies. Therefore we only use one primer, going in one direction. Furthermore, ddNTPs are included in the mixture. ddNTPs are fluorescent nucleotides, a different color per base. These randomly attach and terminate the copying along a particular strand. For an excellent animation of a sequencing reaction, see the Dolan DNA Learning Center. Sequencing, like PCR, is done in a thermalcycler.

1. Add 1 μl of each gene cleaned DNA to a 0.2 ml tube.

2. Prepare primer cocktail:

            In making the cocktail, multiply volumes by the number of samples.

Recipe: 1 μl of primer

2 μl of Big Dye (contains dNTPs,    ddNTPs, enzymes)

                                        6 μl sterile distilled water

3. Add 4 μl of primer cocktail to each tube.

4. Place tubes in the thermalcycler. Set conditions according to primer annealing requirements.