Table 4.
GT RT-qPCR test kit formulation.
| Component | Stock | Volume (μL) | Final concentration |
|---|---|---|---|
| Template | Quantitative Synthetic SARS-CoV-2 RNA: ORF, E, N (ATCC® VR-3276SD™) | 0.5–5 | 5–50,000 copies |
| or | |||
| Full-length viral RNA + HEK293T total RNA | 5 | 5–50,000 copies viral RNA + 0.02–200 ng HEK293T total RNA | |
| Primer/probe: | GT singleplex or multiplex primer/probe mix | 1.5 | 500 nM primers 125 nM probes |
| GT-Master mix: | 5X buffer: 250 mM Tris pH 8.3, 375 mM KCl, 15 mM MgCl2, 47.5% trehalose |
4.0 | 1X buffer: 50 mM Tris pH 8.3, 75 mM KCl, 3 mM MgCl2, 9.5% trehalose |
| 10 mM dNTP | 0.8 | 400 μM | |
| 100 mM DTT | 1.0 | 5 mM | |
| GT-rRI2 | 1.0 | 50 μg/mL | |
| GT-His-Taq2 | 1.0 | 7.5 μg/mL | |
| GT-MMLV2 | 0.5 | 3.3 μg/mL | |
| 20 mg/mL BSA | 1.0 | 1 mg/mL | |
| 25 μM ROX | 0.4 | 500 nM | |
| Water: | Molecular biology grade water | xx | |
| Total volume | 20 |
1
Can be prepared as a 2X mix and stored at −20°C.
2
See Experimental Procedures. Concentration or volume should be adjusted for activity.