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[3H] Reverse transcriptase enzyme assay
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Assay concept>
1536-well assay protocol>
Reagent usage in 96-, 384- and 1536-well formats>
Typical data>
Ordering information>
Assay concept
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1536-well assay protocol
1. | 2 µl reaction mix prepared as follows:
1.56 µl (1.56 µCi) [3H]TTP (TRK576)
2.5 µl (10.7 mM) dNTPs,
Make up to 74 µl with assay buffer |
2. | 1 µl (9 µg/µl) primer/template |
3. | 1 µl assay buffer (50 mM Tris HCl pH8, 80 mM KCl, 10 mM MgCl2 10 mM DTT, 0.05% w/v Nonidet P40) or inhibitor sample |
4.
5. | 1 µl enzyme (0.0025 – 0.06 units/ml)
Incubate for 20 minutes at 37oC |
6. | 1.4 µl (0.56 M EDTA, pH 8) stop buffer
1.6 µl (40 mg) streptavidin yttrium oxide beads (RPNQ0271) |
7. | Incubate at room temperature overnight |
8. | Image for 5 minutes |
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Reagent usage in 96-, 384- and 1536-well format
Assay components | 96-well | 384-well | 1536-well |
Assay volume | 150 µl | 50 µl | 8µl |
Bead | 200 µg | 100 µg | 40 µg |
Enzyme amount | 0.1 units | 0.05 units | 0.001 units |
Primer/template | 10 µl | 5 µl | 1 µl |
Primer/template amount | 2 pmoles | 1 pmole | 0.2 pmoles |
dNTPs | 70 µl | 15 µl | 2 µl |
dNTP amount | 7.5 pmoles | 3.75 pmoles | 0.75 pmoles |
[3H]TTP | 0.44 µCi | 0.22 µCi | 0.04 µCi |
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Typical data
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Ordering information
The following products were used in this assay and can be purchased online
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