1. For double-stranded DNA templates: a. Denature template: 10ml DNA (5-10g or alkaline lysis mini prepDNA) 8ml ddH2O b. Dry-ice precipitate: +10ml 4M NH4OAc 100ml EtOH 2. Annealing reaction:7ml template 2ml Sequenase reaction buffer 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 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. |
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