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, 2 uL each
5.5-2	149 uL, pH 5.5	S-S1, S-S2, S-TFO, 2 uL each
6.5-1	194 uL, pH 6.5	S1, S2, TFO, 2 uL each
6.5-2	194 uL, pH 6.5	S-S1, S-S2, S-TFO, 2 uL each
7.0-1	194 uL, pH 7.0	S1, S2, TFO, 2 uL each
7.0-2	194 uL, pH 7.0	S-S1, S-S2, S-TFO, 2 uL each
7.5-1	194 uL, pH 7.5	S1, S2, TFO, 2 uL each
7.5-2	194 uL, pH 7.5	S-S1, S-S2, S-TFO, 2 uL each
8.5-1	194 uL, pH 8.5	S1, S2, TFO, 2 uL each
8.5-2	194 uL, pH 8.5	S-S1, S-S2, S-TFO, 2 uL each

• PCR: 37℃ 3h

Fluorescence spectrophotometer

• Sample preparation: dilute each sample to 1%.
• Measurement channels

1. IDA channel: excitation 550 nm; emission 670 nm.
2. IDD channel: excitation 550 nm; emission 570 nm.
3. IAA channel: excitation 650 nm; emission 670 nm.