DNA Reaction - Sequencing with Sequenase
1. Following primer annealing, add to the side of the tube in this order:
10 µL Annealing Mix
1 µmL DTT reagent
2 µL Labeling Mix, diluted 1:20 (plasmid) or 1:100 (PCR)
2 µL Sequenase, diluted 1:8.
1 µL α-32P-dATP
2. Pulse reaction in a microfuge.
3. Add 2.5 µL of each ddNTP mix to the wall of the BOTTOM portion of the appropriate tubes, usually set up in the following grid:
G A T C
Reaction 1 -> O O O O
Reaction 2 -> O O O O
Reaction 3 -> O O O O
Reaction 4 -> O O O O
4. Remove Reaction tubes from the microfuge and dispense 3.6 mL aliquots of each reaction mix to the wall of the TOP portion of each of the four termination tubes.
5. Pulse reaction in the microfuge. Incubate tubes at 37° for 10 min.
6. Add 5 µL of the Stop Buffer to each tube. Pulse in the microfuge.
7. Heat tubes to 80° for 3 min. Place on ice.
8. Load 2 µL of each reaction to a sequencing gel in the order G A T C.