Understand the GoTaq 1-Step RT-qPCR System's purpose for sensitive RNA quantification. Program and pre-warm the thermal cycler according to the technical manual (TM355) available at promega.com.
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2
Thaw and prepare reagent components
Place GoTaq one-step RT-qPCR system components, RNA templates, and primers on ice. Visually inspect for complete thawing, then mix immediately by swirling, inversion, or low-speed vortexing to avoid bubble formation.
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3
Assemble reaction mixture in tube
Combine appropriate volumes of RNA, primers, and GoTaq one-step system components in a non-stick sterile tube on ice. Thoroughly mix by pipetting or gentle vortexing between each addition.
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4
Distribute reaction mixture to plate
Carefully distribute reaction volumes to replicate wells of the qPCR plate on ice, avoiding cross-contamination. Include appropriate positive and negative controls for your experiment.
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5
Seal plate and eliminate air bubbles
Cover the plate wells with an optical plate seal. Centrifuge at room temperature for up to 1 minute to eliminate air bubbles and collect reaction volumes at the bottom of each well.
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6
Protect reactions and load thermal cycler
Keep the sealed reactions chilled on ice or in a cold block, protecting them from direct intense light. Place the reaction plate into the pre-warmed and programmed thermal cycler and start the run immediately.
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