Protocol 2
Protocol
Triplex DNA pH Test
- Stock Solution: Dissolve in ultrapure water to 1 mM and store at −20 °C.
- Buffer: 0.5X TBE with 10 mM MgCl2 (pH: 5.5, 6.5, 7.0, 7.5, 8.5)
- Incubation: Dilute the stock with ultrapure water to 1% (10 µM). Add 4 µL each of TFO, S1, and S2 (or S‑TFO, S‑S1, S‑S2) to 388 ul buffers of different pH and incubate at 37 °C for 3 h.
| pH (388 uL) | SsDNA 1 (Sy3) — 4 uL | SsDNA 2 (Sy5) — 4 uL | SsDNA 3 — 4 uL |
|---|---|---|---|
| 5.5 | TFO | S1 | S2 |
| 5.5 | S-TFO | S-S1 | S-S2 |
| 6.5 | TFO | S1 | S2 |
| 6.5 | S-TFO | S-S1 | S-S2 |
| 7.0 | TFO | S1 | S2 |
| 7.0 | S-TFO | S-S1 | S-S2 |
| 7.5 | TFO | S1 | S2 |
| 7.5 | S-TFO | S-S1 | S-S2 |
| 8.5 | TFO | S1 | S2 |
| 8.5 | S-TFO | S-S1 | S-S2 |
Fluorometer
- Donor-only:Cy3-only
- Acceptor-only:Cy5-only
- 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.
Formuals
- α = mean(IDA_Donly / IDD_Donly)--(donor → acceptor leakage ratio)
- β = mean(IDA_Aonly / IAA_Aonly)--(acceptor direct‑excitation by the donor excitation ratio)
- IDA_corr = IDA_meas - α*IDD_meas − β*IAA_meas--(If the result < 0, set it to 0 .)
- Eapp = IDA_corr / (IDA_corr + γ * IDD_meas), here γ ≈ 1-- (approximate)