Synthesize Second Strand cDNA

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This step removes the RNA template and synthesizes a replacement strand to generate blunt-ended, double-stranded cDNA fragments. In place of deoxythymidine triphosphate (dTTP), deoxyuridine triphosphate (dUTP) is incorporated to quench the second strand during amplification and achieve strand specificity.

• RSB (Resuspension Buffer)
• SMM (Second Strand Marking Master Mix)
• AMPure XP (Agencourt AMPure XP)
• Freshly prepared 80% ethanol (EtOH)
• 96-well PCR plate, semiskirted
• Microseal 'B' adhesive film

• Prepare 80% EtOH fresh and discard after one day. The protocol has three cleanup steps that require fresh 80% EtOH.

1. Prepare 80% EtOH from absolute EtOH.
2. Save the following SSS program on the thermal cycler:
• Choose the preheat lid option and set to 40°C
• Reaction volume is 50 µl
• 16°C for 1 hour
• Hold at 4°C for ≤ 16 hours

Generate cDNA

1. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. Add 25 µl SMM to each well.
3. Pipette 10 times, and then seal.
4. Place on the preprogrammed thermal cycler and run the SSS program.

Total program time is ~1 hour and each well contains a volume of 50 µl.

Clean Up cDNA

1. Centrifuge the sealed PCR plate at 280 × g for 10 seconds.
2. Vortex AMPure XP to resuspend.
3. Add 90 µl AMPure XP to each well.
4. Mix using either method:
• Seal and shake at 2000 rpm for 1 minute, and then centrifuge at 280 × g for 10 seconds.
• Slowly pipette until the beads are resuspended.
5. Incubate at room temperature for 5 minutes.
6. Place on the magnetic stand and wait 5 minutes.
7. Remove and discard 130 μl supernatant.
8. Wash beads as follows.
a. Keep on the magnetic stand and add 175 µl fresh 80% EtOH to each well.
b. Wait 30 seconds.
c. Without disturbing the beads, remove and discard all supernatant.
9. Wash beads a second time.
10. With a 20 µl pipette, remove all residual EtOH.
11. Air-dry on the magnetic stand for 2 minutes. Do not overdry the beads.
12. Remove from the magnetic stand.
13. Add 19.5 µl RSB to each well.
14. Mix using either method:
• Seal and shake at 2200 rpm for 1 minute.
• Slowly pipette until the beads are resuspended, and then seal.
15. If shaking did not fully resuspend the beads, slowly pipette until the beads are resuspended, and then seal.
16. Incubate at room temperature for 2 minutes.
17. Centrifuge at 280 × g for 10 seconds.
18. Place on the magnetic stand and wait 2 minutes.
19. Transfer 17.5 µl supernatant to the corresponding well of a new PCR plate.

Small amounts of bead carryover do not affect performance.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at ‑25°C to ‑15°C for up to 7 days.