Contributors: Tank
Purpose
Repeat of Run 1 with a corrected standard series; volume series 10 / 17 / 50 / 83 µl. Test ID 265. Gain 1000, bottom read, 2 min × 670 cycles = 22.3 h, temperature not set. Change made: 4-MU diluted in DMSO this time, not PBS.
Results
| Observation | Value |
|---|---|
| Standard curve now well behaved | 3.125 µM → 2,067 FSU/µM; 6.25 → 2,063; 12.5 → 1,972 (−5%); 25 → 1,754 (−15%) |
| Linear limit established | ≤ 6.25 µM |
| Matrix effect of pure BugBuster | R = 1.33 at 3.125 µM — BugBuster enhances, doesn’t quench |
| Saturated plateau differs between matrices | PBS 137,687 vs BugBuster 123,704 → ~10% genuine quantum-yield quenching |
| Signal decay over 22 h in BugBuster | up to 40%, worse at low concentration; PBS only 5–9% |
| Volume-series rates | 10 µl 34.7 · 17 µl 44.0 · 50 µl 91.0 · 83 µl 119.8 FSU/min → efficiency 100 / 74 / 52 / 42% |
Problems and actions
- Efficiency still falls with volume → cause still unclear at this point (optical artefact vs genuine inhibition — resolved in Run 3).
- Endpoint reading invalid in BugBuster → up to 40% decay over 22 h means only initial rates are usable.
- Still a 22 h read → shorten to ~150 min.