Protocol 2: Difference between revisions
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== Protocol == | == Protocol == | ||
=== Triplex DNA pH Test=== | === Triplex DNA pH Test=== | ||
*'''Stock Solution''': 1 mM | *'''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) | *'''Buffer''': 0.5X TBE with 10 mM MgCl2 (pH: 5.5, 6.5, 7.0, 7.5, 8.5) | ||
*'''Incubation''': | *'''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. | ||
{| class="wikitable"style="text-align:center; width:80%" | {| class="wikitable"style="text-align:center; width:80%" | ||
! T-S1 | ! T-S1 | ||
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| 5.5 , 6.5 , 7.0 , 7.5 , 8.5 | | 5.5 , 6.5 , 7.0 , 7.5 , 8.5 | ||
|} | |} | ||
=== Fluorometer === | === Fluorometer === | ||
*Donor-only:Cy3-only | *Donor-only:Cy3-only | ||
Revision as of 03:32, 15 December 2025
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.
| 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
- 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)