ReadiScript™ RT Reverse Transcription Kit

Allow the ReadiScript™ RT Reaction Mix (5X) to thaw on ice. Before using, vortex reaction mix thoroughly.

The following protocol can be used as a guideline.

1. Prepare the cDNA synthesis reaction by adding the following components to a microcentrifuge tube in the order they appear below:

   Table 1. Reagents composition per well for each reaction.

   Components	Volume (20 µL/reaction)
   ReadiScript™ RT Reaction Mix (5X)	5 µL
   RNA	1-5 µL (Up to 1 µg)
   RNase-Free Water	Up to 20 µL

    

2. Carefully mix the reagents with a gentle vortex followed by a brief centrifuge.

3. Incubate the reaction in a thermal cycler at 65°C for 5 minutes.

4. Cool the reaction at room temperature to allow the primers to anneal to the DNA (approximately 5 minutes).

5. Add 1 µL of ReadiScript™ RT Enzyme to the reaction.

6. Carefully mix the reagents with a gentle vortex followed by a brief centrifuge.

7. Incubate the reaction in a thermal cycler at 25°C for 5 minutes.

8. Incubate the reaction in a thermal cycler at 42°C for 60 minutes.

9. Terminate the reaction by incubating the reaction at 65°C for 10 minutes.

10. Place the completed cDNA synthesis reaction on ice for use in downstream applications. For long-term storage, the reaction can be stored at -20 °C.