XIE/2:2025/Dec: Difference between revisions
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<div style="color: black !important; font-family: 'Times New Roman', SimSun, serif !important, line-height:1.5;"> | <div style="color: black !important; font-family: 'Times New Roman', SimSun, serif !important, line-height:1.5;"> | ||
== 12.12 == | == 12.12 == | ||
*<big>'''<span style="color:red;">Triplex DNA pH Test</span>'''<big> | *<big>'''<span style="color:red;">Triplex DNA pH Test</span>'''</big> | ||
=== Sample preparation === | === 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. | *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. | ||
| Line 10: | Line 10: | ||
#Group 1: add S1, S2, and TFO , 2 µL each. | #Group 1: add S1, S2, and TFO , 2 µL each. | ||
#Group 2: add S-S1, S-S2, and S-TFO , 2 µL each. | #Group 2: add S-S1, S-S2, and S-TFO , 2 µL each. | ||
{| class="wikitable"style="width:80%;" | |||
! 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 | *PCR: 37℃ 3h | ||
=== | === Fluorescence spectrophotometer === | ||
* | *Sample preparation: dilute each sample to 1%. | ||
* | *Measurement channels | ||
#IDA | #IDA channel: excitation 550 nm; emission 670 nm. | ||
#IDD | #IDD channel: excitation 550 nm; emission 570 nm. | ||
#IAA | #IAA channel: excitation 650 nm; emission 670 nm. | ||
Revision as of 08:58, 13 December 2025
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.
- Group 1: add S1, S2, and TFO , 2 µL each.
- 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
- IDA channel: excitation 550 nm; emission 670 nm.
- IDD channel: excitation 550 nm; emission 570 nm.
- IAA channel: excitation 650 nm; emission 670 nm.