Assess cDNA Quality

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This step uses the remaining elution volume (2 µl) from the magnetic bead pellet to assess cDNA QC.

Preparation

Prepare the following consumables:

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0.5X PWB1—Multiply each of the following volumes by the number of samples.

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PWB1 (4 µl)

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Nuclease-free water (4 µl)

These volumes produce 8 µl 0.5X PWB1 per sample.

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PMM4—Pipette to mix, and then centrifuge briefly.

•

WTAP—Pipette to mix, and then centrifuge briefly.

Save the following QC Amplification program on the thermal cycler:

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Set the lid temperature to 105°C

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95°C for 3 minutes

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X cycles of:

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98°C for 15 seconds

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69°C for 4 minutes

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72°C for 5 minutes

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Hold at 4°C

Adjust the program to optimize for different input quality and concentration.

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

Sample Input	Number of QC PCR Cycles (X)
High RNA sample types (eg, cell lines)	10
Low RNA sample types (eg, primary cells and nuclei)	13

Procedure

Keep on the magnetic stand and add 9 µl nuclease-free water.

Without disturbing the pellet, pipette to mix.

Incubate at room temperature for 1 minute.

Without disturbing the beads, transfer 10 µl from the sample tube to a new PCR tube strip.

Place the sample on ice.

Combine the following volumes to prepare QC Master Mix:

Reagent	Volume Per Reaction (μl)	4.4X Reaction Master Mix (µl)	8.8X Reaction Master Mix (µl)
PMM4	6.25	27.5	55
WTAP (undiluted)	1	4.4	8.8
0.5X PWB1	7.75	34.1	68.2
Total	15	66	132

Vortex to mix, and then centrifuge briefly.

Add 15 μl QC Master Mix to each sample.

The total volume per sample is 25 μl.

Pulse vortex and then centrifuge briefly.

10.

Place on the preprogrammed thermal cycler and run the QC Amplification program.

SAFE STOPPING POINT

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