ABI-377 Dye Terminator Sequencing

Dominik Haudenschild, May 7, 97

Keep Reactions in dark whenever possible

DNA 10µl in Water (250-500ng plasmid)

Primer 2µl total of 3.2pmol

Rxn Mix 8µl (Master Mix of RxnMix + Primer is OK)

20µl

Run PCR program:

1: 95° for 0:10

2: 50° for 0:05

3: 60° for 4:00

4: Goto 1 24 more times

5: 4° for ever

6: END (Store frozen until ready to run)

Clean up Dye Terminators by Precipitation

To 0.65 ml tube add:

2.0µl 3M Sodium Acetate pH 4.6-5.2

50µl 95% Ethanol

Add entire 20µl sequencing reaction to 1.5ml tube

Centrifuge 15-30 minutes max rpm

Aspirate supernatant

Rinse with 250µl 70% Ethanol

Centrifuge 5 minutes

Aspirate supernatant

Dry pellet

Clean up Dye Terminators

1ml Sephadex G-50 (Fine) in Water or 0.1x TE

Spin ~3000 RPM for 5 minutes, discard flowthru

Add sequencing reaction (50µl Volume)

Spin ~4000 RPM for 5 minutes

Speed-vac (no heat) flowthru until dry

Resuspend in 10µl of loading buffer

Freeze until ready to run on gel

Make Gel

Clean with WATER ONLY

Dry with KimWipes

Blow away any dust or fibers

Pour, allow to polymerize >1 hour