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文献和实验(12 l) 4 units enzyme (1tube) Mix DNA and Enzyme mix Add 4 ul to 1 ul nucleotides (see nucleotide mixes). Incubate 50-60℃ 15 min. Add 1 ul chase (0.5mM nucleotides) Incubate 15 min. Stop and Load Reaction buffer (5X
one-step reaction (25).Each extension reaction contained 500-750ng of Biomek isolated single-stranded DNA, reaction buffer, nucleotide extension mix, oligonucleotide primer (typically M13 (-40) universal sequencing primer, see Appendix D), either [a-32-P
) 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
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