Amplify cDNA

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• 4X PCR Master Mix
• WTA Primer
• 1.5 ml Safe-Lock PCR Clean tubes
1. Prepare the following consumables:
• 4X PCR Master Mix—Pipette to mix, and then centrifuge briefly.
• WTA Primer—Pipette to mix, and then centrifuge briefly.
2. Save the following cDNA Amplification program on the thermal cycler:
• Set the lid temperature to 105°C
• 95°C for 3 minutes
• 5 cycles of:
• 98°C for 15 seconds
• 69°C for 10 minutes
• 72°C for 5 minutes
• Hold at 4°C

Annealing and extension both occur at 69°C in this profile.

The specified program parameters are critical for assay performance. Do not modify the PCR cycle number. Altering the program causes unusable results.

1. Combine the following volumes in a 1.5 ml tube to prepare a 1:10 dilution of WTA Primer:

Reagent

4.4X Reaction Master Mix (µl)

8.8X Reaction Master Mix (µl)

WTA Primer

3

6

Nuclease-free water

27

54

Total

30

160
2. Combine the following volumes to prepare Whole Transcriptome Amplification (WTA) Master Mix:

Reagent

Volume Per Reaction (μl)

4.4X Reaction Master Mix (µl)

8.8X Reaction Master Mix (µl)

4X PCR Master Mix

15

66

132

Diluted WTA Primer

6

26.4

52.8

Total

21

92.4

184.8
3. Vortex to mix, and then centrifuge briefly.
4. Add 21 µl WTA Master Mix to each sample tube.
5. Pulse vortex for 5 seconds in the tube stand, and then centrifuge briefly.

If PIPs are pelleted in the centrifuge, repeat vortexing and centrifuge steps.

6. Place on the preprogrammed thermal cycler and run the cDNA Amplification program.

SAFE STOPPING POINT

If you are stopping, leave the sample tubes on the thermal cycler at 4°C or store samples at 2°C to 8°C overnight.