Check Samples

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• QCP
• QCT
• qPCR master mix containing green fluorescent dye
• Nuclease-free water
• Disposable reagent trough
• Microcentrifuge tubes, 1.7 ml
• qPCR plates
• Pipette tips, 10 µl
• Pipette tips, 20 µl
• Pipette tips, 1000 µl
1. Measure the concentration of DNA extracted from FFPE samples using PicoGreen or similar fluorescent dye assays. Dilute the DNA to a concentration of 1 ng/μl.
2. Prepare the following consumables:
• QCP—Vortex to mix.
• QCT—Vortex to mix.
3. Create 10 μl aliquots of QCT in tubes labeled QCT-ST.

If you are using QCT for multiple PCR runs, store the tubes at -25°C to -15°C. Use a fresh QCT aliquot for each PCR run. The original box accommodates a minimum of six aliquots.

4. Save the following qPCR program on the qPCR instrument:
• Hot start (if required by master mix manufacturer):
• 50°C for 2 minutes
• 95°C for 10 minutes
• 40 cycles of:
• 95°C for 30 seconds
• 57°C for 30 seconds
• 72°C for 30 seconds
1. Add 990 μl of nuclease-free water into a fresh 10 μl aliquot of QCT to create a 100-fold dilution of QCT.
2. Vortex briefly to mix, and then centrifuge briefly at 280 × g.
3. For 10 µl reaction volumes, prepare the plate as follows.
a. Add 2 µl 100-fold diluted QCT from the QCT-ST tube into three wells of the qPCR plate.
b. Add 2 μl genomic DNA (1 ng/μl) from FFPE samples into three wells of the qPCR plate.
c. Add 2 μl nuclease-free water into three wells of the qPCR plate for NTC.
4. For 20 µl reaction volumes, prepare the plate as follows.
a. Add 4 μl 100-fold diluted QCT from the QCT-ST tube into three wells of the qPCR plate.
b. Add 4 μl genomic DNA into three wells of the qPCR plate.
c. Add 4 μl nuclease-free water into three wells of the qPCR plate for NTC.
5. To prepare the qPCR premix, combine the following volumes. Multiply each volume by the number of samples or controls.

Component

10 µl reaction volumes (μl)

20 µl reaction volumes (μl)

qPCR master mix

5

10

QCP

0.8

1.6

Nuclease-free water

2.2

4.4

Total volume per well

8

16

For example, if analyzing 100 DNA samples in a 384-well plate, prepare the qPCR premix for 337 replicates (306 replicates + 10% overfill). Calculate the final volume as follows.

337 x 8 μl qPCR premix = 2.696 ml

6. Invert the qPCR premix container 10 times to mix. Tap the container on the bench or other hard surface to collect contents.
7. Transfer the qPCR premix into a clean trough.
8. Dispense the appropriate quantity of qPCR premix into each well containing gDNA, QCT, and NTC.
• For 10 μl final volume, dispense 8 μl qPCR premix.
• For 20 μl final volume, dispense 16 μl qPCR premix.

The volume specified is critical to the success of the assay results.

9. Seal the plate according to manufacturer instructions and centrifuge briefly at 280 × g.
10. Make sure that the optical seal is clean from any liquid or dust. Place the plate in the qPCR instrument in the correct orientation.
11. Run the preprogrammed qPCR program.