Amplify and Tag Targets

This process amplifies target regions to create two separate libraries per sample using two primer mixes: Mix A using Primer Mix A and Mix B using Primer Mix B.

• TTM (TruSight Tumor Targeting Mix)
• TPA (TruSight Tumor Primer Mix A)
• TPB (TruSight Tumor Primer Mix B)
• TTE (TruSight Tumor Targeting Enzyme)
• Sample DNA
• RNase/DNase-free water
• 1.7 ml microcentrifuge tubes (2)
• 96-well PCR plate
• Microseal 'A' film
• Microseal 'B' adhesive seal

Use Microseal 'A' when sealing the plate before placing it on the thermal cycler. Use Microseal 'B' for other steps that require a sealed plate.

1. Prepare the following consumables:

PCR Component

Per Well

Per 8 Samples

TTM

-25°C to ‑15°C

Thaw at room temperature. Vortex each tube to mix. Centrifuge briefly.

TPA

-25°C to ‑15°C

Thaw at room temperature. Vortex each tube to mix. Centrifuge briefly.

TPB

-25°C to ‑15°C

Thaw at room temperature. Vortex each tube to mix. Centrifuge briefly.

TTE

-25°C to ‑15°C

Centrifuge briefly and place on ice or in an enzyme cooler.

Sample DNA

-25°C to ‑15°C

Thaw at room temperature.
2. Label two 1.7 ml microcentrifuge tubes as Master Mix A and Master Mix B.
3. Save the following program as TST15 PCR1 on a thermal cycler with a heated lid:
a. Choose the preheated lid option and set it to 102°C.
b. Set the reaction volume to 15 μl.
c. Set to 98°C for 3 minutes.
d. Set 16 cycles of the following temperatures and times:
• 96°C for 45 seconds
• 70°C for 1 minute
• 54°C for 3 minutes with the following ramp rate, depending on the thermal cycler:

Thermal Cycler

Ramp Rate

Bio-Rad C1000 Thermal Cycler

Down

0.1 °C/s

Up

0.1 °C/s

Bio-Rad S1000 Thermal Cycler

Down

0.1 °C/s

Up

0.1 °C/s

Applied Biosystems GeneAmp PCR System 9700

Down

7%

Up

7%

Applied Biosystems Veriti Thermal Cycler

Down

4.5%

Up

4.0%

Eppendorf Mastercycler ep Gradient

Down

6%

Up

4%

Eppendorf Mastercycler ep Gradient‑S

Down

2%

Up

2%
• 72°C for 15 seconds with the following ramp rate, depending on the thermal cycler:

Thermal Cycler

Ramp Rate

Bio-Rad C1000 Thermal Cycler

Down

0.1 °C/s

Up

0.1 °C/s

Bio-Rad S1000 Thermal Cycler

Down

0.1 °C/s

Up

0.1 °C/s

Applied Biosystems GeneAmp PCR System 9700

Down

7%

Up

7%

Applied Biosystems Veriti Thermal Cycler

Down

4.5%

Up

4.0%

Eppendorf Mastercycler ep Gradient

Down

6%

Up

4%

Eppendorf Mastercycler ep Gradient‑S

Down

2%

Up

2%
• 72°C for 5 minutes
• Hold at 10°C

Ramp rate is critical for TruSight Tumor 15 performance. Failure to set the ramp rate results in low product yields and high primer-dimer.

1. Quantify the sample DNA using a fluorometric method specific for double-stranded DNA, such as AccuClear (recommended), Qubit, or PicoGreen.
2. Dilute each sample DNA to 2 ng/µl with RNase/DNase-free water in a final volume of 12.5 µl.
3. Combine the following reagents in separate microcentrifuge tubes to create PCR master mixes for TPA and TPB.

Prepare a minimum of 4 samples.

PCR Component

Per Well

Per 8 Samples

Per 16 Samples

Per 24 Samples

TTM

5.875 µl

47 µl

94 µl

141 µl

TPA or TPB

6.25 µl

50 µl

100 µl

150 µl
4. Pipette to mix.
5. Add 10 µl of the following PCR master mixes to each well:
• Master Mix A—Rows A and C
• Master Mix B—Rows B and D
6. Add 5 µl of 2 ng/μl DNA to the corresponding well.
• Samples 1–12—Rows A and B
• Samples 13–24—Rows C and D
7. Pipette to mix.
8. Apply the seal and centrifuge at 1000 × g for 1 minute.
9. Immediately place on a thermal cycler and run the TST15 PCR1 program.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at 2°C to 8°C for up to 3 days. Alternatively, leave on the thermal cycler overnight.