Protocol 2

Protocol

Triplex DNA pH Test

• Stock Solution: 1 mM

• Buffer: 0.5X TBE with 10 mM MgCl2 (pH: 5.5, 6.5, 7.0, 7.5, 8.5)

• Incubation: 37℃;C, 3 h

T-S1	T-S2	TFO	Buffer	pH
4 μL	4 μL	4 μL	388 μL	5.5 , 6.5 , 7.0 , 7.5 , 8.5

Fluorometer

• Donor-only:Cy3-only
• Acceptor-only:Cy5-only
• 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.

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)