Thermo Fisher Scientific TaqMan Quick Reference

QUICK REFERENCE

TaqMan™ SCID/SMA Assay

C

atalog Numbers A47927, A47928, A47929

Pub. No. MAN0018922 Rev. C.0

Note: For safety and biohazard guidelines, see the “Safety” appendix in the TaqMan™ SCID/SMA Assay User Guide
(Pub. No. MAN0018921). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves.

This Quick Reference is intended as a benchtop reference for experienced users of the TaqMan™ SCID/SMA Assay. For detailed
instructions and ordering information for additional products, see the TaqMan™ SCID/SMA Assay User Guide (Pub. No. MAN0018921).

Contents and storage

Table 1 TaqMan™ SCID/SMA Assay

Cat. No. Number of reactions Storage

A47927 4,000

A47928 8,000

A47929 20,000

• 2–8°C for up to 3 months

−30°C to −10°C for long‑term storage

•

Procedural guidelines

•

Keep the PCR Reaction Mix on a chilled block during real-time PCR reaction setup.

• The PCR Reaction Mix can be formulated up to 48 hours before sample preparation and stored at 2–8°C. See “Prepare the real-time
PCR reactions” on page 2.

• Ensure that the instrument is calibrated for each detector dye and passive reference dye, according to the instrument user guide. For
more information, see the TaqMan™ SCID/SMA Assay Calibration Guide (Pub. No. MAN0019378).

Before you begin

DBS wash buer and stabilizing buer are stable at room temperature and can be prepared several days in advance. They do not need to
be prepared fresh.

• Prepare DBS wash buer—add 5 g of liquid Thesit™ reagent into 100 mL of luke warm 1X PBS to prepare a 5% stock, then perform
serial dilution using luke warm 1X PBS to prepare 0.5% DBS wash buer.

DBS wash buer is 1X PBS and 0.5% (w/v) Thesit™ reagent.

Note:

Alternatively, a higher or lower concentration and/or volume of stock can be prepared based on the throughput of your laboratory.

·

Heat the Thesit™ reagent until liquid and the 1X PBS until luke warm to allow the Thesit™ reagent to dissolve readily.

·

• Prepare stabilizing buer—add 0.5 mL of 10% Tween™-20 Surfact-Amps™ Detergent Solution to 100 mL of TE Buer.

Stabilizing buer is modified TE buer (10 mM Tris, 0.1 mM EDTA, pH 8) with 0.05% Tween™-20 detergent added.

Extract the DNA

1. Place a DBS punch (1.5 mm or 3.2 mm) from a blood card into the well of the 0.2 mL optical 96-well reaction plate.

Note: Use only one punch per well.

For Research Use Only. Not for use in diagnostic procedures.

2. Wash the DBS punch.

a. Add the DBS wash buer to each sample well.

• 1.5 mm DBS punch: 70 µL

• 3.2 mm DBS punch: 150 µL

b. Seal the plate with a clear adhesive film, then centrifuge at 2400 rpm for 1 minute to wet and submerge the punch.

c. Shake the plate for 30 minutes at 1500 rpm on a microplate shaker.

d. Centrifuge briefly to collect the contents at the bottom of the wells, then remove and discard the supernatant.

e. Add 150 µL of water to each sample well, then remove and discard the water.

3. Add 5 µL of DNA Extract All Reagents Kit Lysis Solution to each sample well.

4. Seal the plate, then centrifuge briefly to collect the contents at the bottom of the wells.

5. Incubate the reaction plate for 5 minutes at 95°C.

6. Centrifuge briefly to collect any condensed droplets at the bottom of the wells, then cool to room temperature before opening the

seal.

Note: Optionally, place the plate on a chilled block to quickly cool the plate.

7. Add 35 µL of stabilizing buer to each sample well.

Note: The total lysate volume per 1.5 mm or 3.2 mm DBS punch is 40 µL.

8. Seal the plate, vortex briefly, then centrifuge briefly to collect the contents at the bottom of the wells.

9. Transfer the lysate to a fresh optical reaction plate according to the following table.

DBS punch Lysate volume per well Reaction plate

1.5 mm 9 µL 96-well (0.1 mL or 0.2 mL)

3.2 mm 9 µL 96-well (0.1 mL or 0.2 mL)

3.2 mm 6.75 µL 384-well

Prepare the real-time PCR reactions

1. P

repare the PCR Reaction Mix in an appropriately sized microcentrifuge tube according to the following table.

Component

2X TaqPath™ P

20X TaqMan™ SCID/SMA Assay 1 µL 0.75 µL

Total PCR Reaction Mix volume 11 µL 8.25 µL

[1]

A

dd 10% overage for pipetting loss. Excess PCR Reaction Mix can be stored at 2–8°C for up to 48 hours.

roAmp™ Multiplex Master Mix 10 µL 7.5 µL

96-well plate 384-well plate

Volume per reaction

2. Vortex to mix, then centrifuge briefly to collect the contents at the bottom of the tube.

3. Add the PCR Reaction Mix to each well containing the transferred lysate.

[1]

• 96-well plate: 11 µL

• 384-well plate: 8.25 µL

4. Seal the plate with an optical adhesive film, vortex to mix, then centrifuge briefly to collect the contents at the bottom of the wells.

2 T

aqMan™ SCID/SMA Assay Quick Reference