Thermo Fisher Scientific TaqMan SCID/SMA Quick Reference

QUICK REFERENCE

TaqMan™ SCID/SMA Plus Assay

C

atalog Numbers A48566, A48567, A48568, A48569

Pub. No. MAN0019380 Rev. A.0

Note: For safety and biohazard guidelines, see the “Safety” appendix in the TaqMan™ SCID/SMA Plus Assay User Guide
(Pub. No. MAN0019381). 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 Plus Assay. For
detailed instructions and ordering information for additional products, see the TaqMan™ SCID/SMA Plus Assay User Guide (Pub.
No. MAN0019381).

Contents and storage

Table 1 TaqMan™ SCID/SMA Plus Assay

Cat. No.

[1]

A48566 1,000

Number of reactions Storage

A48567 4,000

A48568 8,000

A48569 20,000

[1]

C

atalog numbers that appear as links open the web pages for those products.

• 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 step 1 in “Prepare the
real-time PCR reactions” on page 3.

• The assembled PCR Reaction Mix containing the sample lysate is stable for at least 48 hours when stored at 2–8°C. See step 3 in
“Prepare the real-time PCR reactions” on page 3.

• 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 Plus Assay Calibration Guide (Pub. No. MAN0019382).

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—to prepare a 5% stock, add 5 g of liquid Thesit™ reagent into lukewarm 1X PBS to a final volume of
100 mL. Mix well, then dilute using lukewarm 1X PBS to prepare 0.5% DBS wash buer.

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

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 lukewarm 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.

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

Prepare the standard curve

The plasmids used to prepare the standard curve for TREC and KREC quantitation are as follows.

Target GeneArt™ Construct ID Size in base pairs (bp) Concentration Approximate copies per µL

TREC 18ACJF2P 2747 bp 1 mg/mL 3.37 x 1011 copies/µL

KREC 18ACJFTP 2786 bp 1 mg/mL 3.33 x 1011 copies/µL

1. Dilute the TREC plasmid by 1:10 in prepared stabilizing buer (for example, 5 µL of plasmid in 50 µL total of stabilizing buer—

0.05% Tween®-20 detergent in low TE buer), then denature for 10 minutes at 95°C to linearize the plasmid.

The final concentration of the denatured plasmid will be approximately 3.3 x 1010 copies/µL.

Note: Linearization of the plasmid is required only once.

2. Dilute the denatured plasmid solution by 1:1000 in stabilizing buer to prepare a stock.

The final concentration of the stock will be approximately 3.3 x 107 copies/µL and must be stored at 4°C.

3. Prepare serial dilutions as needed.

All of the serial dilutions must be diluted in stabilizing buer and stored at 4°C.

4. Repeat step 1 to step 3 for the KREC plasmid.

The recommended standard curve points are as follows.

Standard curve Copies per µL of plasmid Approximate copies per reaction

Point 1 3.3 x 105 copies/µL 3.0 x 106 copies/r

Point 2 3.3 x 104 copies/µL 3.0 x 105 copies/reaction

Point 3 3.3 x 103 copies/µL 3.0 x 104 copies/reaction

Point 4 3.3 x 102 copies/µL 3.0 x 103 copies/reaction

Point 5 3.3 x 101 copies/µL 3.0 x 102 copies/reaction

Point 6 3.3 x 100 copies/µL 3.0 x 101 copies/reaction

[1]

W

e recommend running each point in triplicate and using 9 µL of plasmid solution in a 20 µL PCR reaction in a 96-well plate. For a 384-well plate, adjust the plasmid serial dilution

as needed and calculate copies per reaction based on 6.75 µL of plasmid solution in a 15 µL PCR reaction.

eaction

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.

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.

[1]

3. Add 5 µL of DNA Extract All Reagent 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.

2 T

aqMan™ SCID/SMA Plus Assay Quick Reference