Protocol for TotalScript™ RNA-Seq Kit

TotalScript RNA-Seq Kit
2. Incubate each sample for 1 minute at 45°C
3. Incubate each sample for 30 minutes at 37°C
4. Add 1 μl of Gap-Fill Enzyme.
5. Incubate each sample for 30 minutes at 37°C. Then hold at 4°C.
4.D. Purify the reactions using AMPure XP magnetic beads.
1. Prepare at least 500 µl of fresh 80% ethanol solution for each sample
2. Add 25 μl AMPure beads to each reaction. Mix well and let stand at room
temperature for 5 minutes.
3. Place the tubes on magnetic stand and capture beads for 5 minutes, then remove
the unbound material.
4. While still on the magnetic stand, wash the beads twice with 250 μl 80% ethanol.
5. Remove any remaining ethanol and allow the beads to dry for 5 minutes.
6. Remove the tubes from the magnetic stand and resuspend the beads in 25 μl of
Elution Buffer.
7. Place the tubes on the magnetic stand and capture the beads for 5 minutes. The
tagmented cDNA is now in the Elution Buffer phase.
8. Transfer 24 μl of the eluted, gap-filled DNA to fresh PCR tubes and cool on ice.
4.E. PCR amplification
Additionally required (provided by the user)
2X Phusion® High Fidelity PCR Master Mix with HF Buffer (NEB, cat. no. M0531) or
2X Phusion® High Fidelity PCR Master Mix with GC Buffer (NEB, cat. no. M0532)
1. Briefly centrifuge each tube, then combine on ice:
12 µl Gap-Filled cDNA from Step 4.D*
0.5 µl TotalScript PCR Primer Cocktail
12.5 µl 2X Phusion High-Fidelity PCR Master Mix (HF Buffer or GC Buffer)
25 µl total volume
*Store the remaining Gap-Filled cDNA at –20°C.
2. PCR cycle conditions: The number of PCR cycles is dependent on the input RNA and
the cDNA primer used in 1st-strand cDNA synthesis (Part 3.A):
Input Random Primer Mix dT
1 ng 12 14 16
5 ng 10 12 14
Heat at 95°C for 2 minutes.
Then perform the appropriate number of cycles of:
94°C for 10 seconds
60°C for 30 seconds
72°C for 1 minute
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