DNA SEQUENCING (SANGER)

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.