XIE/2:2025/Dec

12.12

• Triplex DNA pH Test

Sample preparation

• ssDNA stocks: s1 (3'Cy5), S2, TFO (3'Cy3), S‑S1 (3'Cy5), S‑S2, and S‑TFO (3'Cy3) were each prepared at 1 mM in nuclease-free water.
• Buffers: PBS adjusted to pH 5.5, 6.5, 7.0, 7.5, and 8.5; prepare 50 mL of each pH condition.

Incubation

• For each pH condition, set up two groups (Group 1 and Group 2). To each reaction, first add 194 µL of the corresponding PBS buffer.

1. Group 1: add S1, S2, and TFO , 2 µL each.
2. Group 2: add S-S1, S-S2, and S-TFO , 2 µL each.

Name	Buffer	SsDNA
5.5-1	194 uL, pH 5.5	S1, S2, TFO 各 2 uL
5.5-2	149 uL, pH 5.5	S‑S1, S‑S2, S‑TFO 各 2 uL
6.5-1	194 uL, pH 6.5	S1, S2, TFO 各 2 uL
6.5-2	194 uL, pH 6.5	S‑S1, S‑S2, S‑TFO 各 2 uL
7.0-1	194 uL, pH 7.0	S1, S2, TFO 各 2 uL
7.0-2	194 uL, pH 7.0	S‑S1, S‑S2, S‑TFO 各 2 uL
7.5-1	194 uL, pH 7.5	S1, S2, TFO 各 2 uL
7.5-2	194 uL, pH 7.5	S‑S1, S‑S2, S‑TFO 各 2 uL
8.5-1	194 uL, pH 8.5	S1, S2, TFO 各 2 uL
8.5-2	194 uL, pH 8.5	S‑S1, S‑S2, S‑TFO 各 2 uL

• PCR: 37℃ 3h

荧光分光光度计

• 样品处理：每个样稀释至1%
• 测量通道

1. IDA-measure:激发波长 550 nm;发射波长 670 nm
2. IDD-measure:激发波长 550 nm;发射波长 570 nm
3. IAA-measure:激发波长 650 nm;发射波长 670 nm