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DNTP |
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Amplification was done in a 50-[micro]L reaction volume containing 10 [micro]L 5x buffer, 2 [micro]L deoxynucleotide triphosphate mix (10 mmol/L), 2 [micro]L enzyme mix (all from QIAGEN), 2 [micro]L RNase inhibitor (Promega, Mannheim, Germany), 1. The incubation with a mixture containing digoxigenin deoxynucleotide triphosphate, unlabeled deoxynucleotide triphosphate, and terminal transferase enzyme was developed in a humidified chamber at 37[degrees]C for 1 hr. A 25-[micro]L reaction was conducted in a LightCycler (Roche, Indianapolis, IN, USA) with 10 mmol/L of each deoxynucleotide triphosphate, 600 nmol/L of each primer, 200 nmol/L of probe, and 3 [micro]L of RNA. |
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