Anneal RNA

Start with this section if using RNA only sample inputs.

During this process, random hexamers are annealed to the extracted RNA to prepare for cDNA synthesis. The random hexamers are contained in EPH3. In most cases, random hexamers are the best choice for first strand cDNA synthesis. There may be cases where different reverse transcriptase (RT) primers, such as target specific primers, may perform better. The random hexamers are contained in EPH3. However, in some cases, other reverse transcriptase (RT) primers— such as target specific primers—may perform better. If random hexamers are not used in the reaction, it will affect total salt concentrations. Carefully analyze the targets and goals of your experiment to determine the best RT primers to use, or consult with the Illumina Technical Support team.

• EPH3 (Elution Prime Fragment 3HC Mix)
• 96-well PCR Plate
• Microseal 'B' adhesive seals
• EPH3—Vortex to mix before each use.
1. Prepare the following consumables:
• EPH3—Vortex to mix.
2. Save the following IMAP Anneal program on the thermal cycler:
• Choose the preheat lid option at 105°C
• Set the reaction volume to 17 µl
• 65°C for 3 minutes
• Hold at 4°C
3. Label a new PCR plate CDNA1.
1. Add 8.5 µl EPH3 to each well.
2. Add 8.5 µl eluted sample to each well.
3. Seal and shake at 1600 rpm for 1 minute.
4. Centrifuge at 1000 × g for 1 minute.
5. Place on the preprogrammed thermal cycler and run the IMAP Anneal program.