1. For double-stranded DNA templates:
a. Denature template: 10ml DNA (5-10g or alkaline lysis mini prepDNA)
8ml ddH2O
2ml 2N NaOH
-incubate 30' at 37℃.
b. Dry-ice precipitate: +10ml 4M NH4OAc
100ml EtOH
-70% EtOH wash
-vacuum dry briefly
-resuspend in 7ml ddH2O
2. Annealing reaction:7ml template
2ml Sequenase reaction buffer
1ml primer (2-3pmol T3, T7, etc.)
-incubate 10' at 65℃
-remove the entire heat block to RT and cool slowly to &30℃
-chill on ice for use in Step 7.
3. While cooling, pipet 2.5ml of each Termination Mix into a 96-well microtiter dish. Use red- capped tubes for dGTP rxns or orange-capped tubes for dITP rxns. Cover the dish with sealing tape and set aside for Steps 6 " 8.
4. Dilute Labeling Mix (green-capped dGTP) 1:5 to working concentration with ddH2O.
5. Dilute Sequenase Version 2.0 1:8 with ice-cold Enzyme Dilution Buffer.
6. Prewarm termination mixes from Step 3 in the 37℃ bath.
7. Labeling reaction: 10ml annealing reaction (Step 2)
1.0ml 0.1M DTT
2.0ml Diluted Labeling Mix
0.5ml [35S]dATP
2.0ml Diluted Sequenase
-mix and incubate 2-5' at RT.
8. Termination reaction: Transfer 3.5ml of the labeling rxn to each termination well, mix and incubate 5' at 37℃.
9. Stop rxns by adding 4ml Stop Solution.
10. Heat samples for 3-5' at 90-100℃ just prior to loading.
