Quick Sequencing Protocol

Dominik Haudenschild 7-18-94, after Sequenase protocol, at CBRC

Alkaline Denaturation

Combine in a small eppendorf tube:
DNA 3-5µg in up to 8µl
1.0M NaOH 2µl
Primer 1µl
H
20 total volume of 11µl

Mix thoroughly, incubate for 37°C for 10 minutes, place on ice

Add to above tube:
1.0M HCl 2µl
Plasmid reaction buffer 2µl (0.4M Tris, 0.1M MgCl2, 0.25M NaCl pH7.5)

Annealing

Incubate primer/template/buffer mixture at 37°C for 10 minutes, then place on ice

During these 10 minutes
-Label, fill and cap tubes with 2.5µl of each Termination mix, (Red capped vials)
-Dilute Labeling mix 5-fold to working concentration with water
Labeling mix 2µl (per sequence)
Water 8µl (per sequence)

Pre-warm 4 termination tubes from step 3 to 37°C

Labeling reaction

To ice-cold annealed DNA mixture 15µl add
DTT (0.1M) 1µl
Diluted Labeling mix 2µl
35S dATP 0.5µl
Diluted Sequenase 2µl
TOTAL 20.5µl

Mix and incubate 5 minutes at room temperature

Termination reaction

Transfer 4.5µl of labeling reaction to each termination tube (G, A, T, and C), mix

Continue Termination reaction at 37°C for 5 minutes

Stop the reactions by adding 4µl of Stop Solution

Make gel

25.2g urea
7.2ml 10x TBE
6ml long ranger
to 60 ml with Milli-Q then 300µl AMPS, 30µl TEMED

Load gel

Heat samples to 75°C immediately before loading onto sequencing gel

Load 2-3µl in each lane