TaqMan™ SCID/SMA Plus Assay

USER GUIDE

Catalog Numbers A48566, A48567, A48568, A48569, A52275

Publication Number MAN0019381
Revision B.0

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

 Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, California 94566 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0019381

Revision Date Description
B.0 11 August 2021 • Updated the TREC Construct ID.
• Added Cat. No. A52275 for 100 reactions.
• Added recommendations regarding plasmid orders.
• Updated real-time PCR reactions preparation to include
information about NTC.

A.0 12 January 2021 New document.

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product,
you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a
registered trademark of Roche Molecular Systems, Inc., used under permission and license. Thesit is a trademark of Desitin Arzneimittel
GmbH. Cy is a registered trademark of GE Healthcare. Tween is a registered trademark of Croda Americas, Inc.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
 Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Plasmid ordering instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ CHAPTER 2 Calibration information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

™ ™
Dye spectral calibration plates for the QuantStudio 6 / QuantStudio 7 Flex
Real-Time PCR System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
™
Dye spectral calibration plates for the QuantStudio 12K Flex Real–Time PCR System . . . 9
™
Dye spectral calibration plates for the QuantStudio 5 Real‑Time PCR System . . . . . . . . 10
™
Dye spectral calibration plates for the QuantStudio Dx Real‑Time PCR
Instrument (RUO mode) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

■ CHAPTER 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Prepare the standard curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Extract the DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Prepare the real-time PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Set up and run the real-time PCR instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Overview of baseline and threshold values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■ APPENDIX A Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ APPENDIX B Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

TaqMan™ SCID/SMA Plus Assay User Guide 3

 1 Product information

■ Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
■ Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
■ Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.

Product description
The Applied Biosystems™ TaqMan™ SCID/SMA Plus Assay is a multiplexed real-time PCR assay that
detects, with high specificity, TREC, KREC, and SMN1. The assay is designed to be used on dried
blood spot (DBS) punches and extracts DNA directly from the punches without the need for a DNA
purification step. It is compatible with QuantStudio™ instruments that have been calibrated for FAM™
dye, VIC™ dye, JUN™ dye, ABY™ dye, and MUSTANG PURPLE™ dye. A 96-well plate and a 384-well
plate workflow are available.
The assay includes the following key features:
• A simple workflow to extract DNA directly from a DBS punch.
• An optimized SMN2 blocker to increase the specificity to SMN1.

The assay has been optimized for high specificity to SMN1 because it can be difficult to accurately
detect the presence of just SMN1, which has high homology with SMN2. In a sample with a
homozygous deletion of SMN1 and in the presence of a high number of SMN2 copies (greater than
what would be biologically expected), the assay still detects the presence of a homozygous SMN1
deletion.

Contents and storage

Cat. No.[1] Number of reactions Storage

A52275 100

A48566 1,000 Follow the recommended storage conditions

listed on each component label.
A48567 4,000
Note: Components A48609 and A48610
A48568 8,000 can be stored at 2–8°C for up to 3 months.

A48569 20,000
[1] Catalog numbers that appear as links open the web pages for those products.

4 TaqMan™ SCID/SMA Plus Assay User Guide

 Chapter 1 Product information
Required materials not supplied 1

Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Catalog numbers that appear as links open the web pages for those products.

Item Source

One of the following Applied Biosystems™ instruments:

• QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System Contact your local sales office.
• QuantStudio™ 12K Flex Real–Time PCR System
• QuantStudio™ 5 Real‑Time PCR System
• QuantStudio™ Dx Real‑Time PCR Instrument (RUO mode)

Or use a compatible real-time PCR instrument from another supplier.

Equipment

Centrifuge with adapter for 96- or 384-well plates MLS

Laboratory mixer (Vortex or equivalent) MLS

Microplate shaker MLS

Microcentrifuge MLS

Pipettors MLS

Cooling block MLS

Plastics and other consumables

Plates and seals for your instrument thermofisher.com/plastics

Disposable gloves MLS

Pipette tips with filters MLS

Polypropylene tubes MLS

Reagents and kits

Tween®-20 Surfact-Amps™ Detergent Solution 28320

TE Buffer 12090015

PBS (1X), pH 7.4 10010049

Thesit™ reagent MLS

Nuclease-free Water (not DEPC-Treated) AM9938

TaqMan™ SCID/SMA Plus Assay User Guide 5

 Chapter 1 Product information
1 Required materials not supplied

Item Source

Plasmids

TREC plasmid (GeneArt™ Construct ID 18ACJFZP) See “Plasmid ordering instructions”

KREC plasmid (GeneArt™ Construct ID 18ACJFTP)

Plasmid ordering instructions

To reorder the TREC plasmid or KREC plasmid, complete the steps as follows:

1. Email [email protected].

2. Request a plasmid "reorder" using the TREC and KREC construct ID.

3. Request 5 μg, lyophilized.

6 TaqMan™ SCID/SMA Plus Assay User Guide

 2 Calibration information

■ Dye spectral calibration plates for the QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time
PCR System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
■ Dye spectral calibration plates for the QuantStudio™ 12K Flex Real–Time PCR System . . . . . . . . . 9
■ Dye spectral calibration plates for the QuantStudio™ 5 Real‑Time PCR System . . . . . . . . . . . . . . 10
■ Dye spectral calibration plates for the QuantStudio™ Dx Real‑Time PCR Instrument
(RUO mode) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Dye spectral calibration plates for the QuantStudio™ 6 /

QuantStudio™ 7 Flex Real-Time PCR System
See your instrument user guide for recommended calibration schedules and detailed calibration
instructions.

Table 1 QuantStudio™ Real‑Time PCR Software v1.6.1 or later

Plate[1] Cat. No.[2]

96-well 0.2 mL

QuantStudio™ 3/5 Spectral Calibration Plate 1

A26331
(FAM™, VIC™, ROX™, and SYBR™ dyes), 96‑Well 0.2‑mL

QuantStudio™ 3/5 Spectral Calibration Plate 2, 96‑Well 0.2‑mL

A26332
(ABY™, JUN™, and MUSTANG PURPLE™ dyes)

96-well 0.1 mL

QuantStudio™ 3/5 Spectral Calibration Plate 1,

A26336
(FAM™, VIC™, ROX™, and SYBR™ dyes), 96‑well, 0.1 mL

QuantStudio™ 3/5 Spectral Calibration Plate 2 (ABY™, JUN™, MUSTANG PURPLE™

A26337
dyes), 96‑well Fast (0.1‑mL) Plate

384-well

QuantStudio™ 5 Spectral Calibration Plate 1, (FAM™, VIC™, ROX™

A26334
TAMRA™, and SYBR™ dyes), 384-well

QuantStudio™ 5 Spectral Calibration Plate 2 (ABY™, JUN™, MUSTANG PURPLE™,

A26335
NED™, and Cy®5 dyes), 384-well
[1] Dye spectral calibration plates for the QuantStudio™ 3 and 5 Instruments are used for the calibration procedure with the
QuantStudio™ Real‑Time PCR Software v1.6.1 or later.
[2] Catalog numbers that appear as links open the web pages for those products.

TaqMan™ SCID/SMA Plus Assay User Guide 7

 Chapter 2 Calibration information
2 Dye spectral calibration plates for the QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System

Table 2 QuantStudio™ Real‑Time PCR Software v1.3

Plate Cat. No.[1]

96-well 0.2 mL

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well A24738

FAM™ Dye Spectral Calibration Plate, 96-well 4432327

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well A24737

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well 4461599

VIC™ Dye Spectral Calibration Plate, 96-well 4432334

96-well 0.1 mL

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well A24734

FAM™ Dye Spectral Calibration Plate, Fast 96-well 4432389

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well A24735

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well 4457328

VIC™ Dye Spectral Calibration Plate, Fast 96-well 4432396

384-well

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well A24736

FAM™ Dye Spectral Calibration Plate, 384-well 4432271

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well A24733

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well 4457334

VIC™ Dye Spectral Calibration Plate, 384-well 4432278

[1] Catalog numbers that appear as links open the web pages for those products.

8 TaqMan™ SCID/SMA Plus Assay User Guide

 Chapter 2 Calibration information
Dye spectral calibration plates for the QuantStudio™ 12K Flex Real–Time PCR System 2

Dye spectral calibration plates for the QuantStudio™ 12K

Flex Real–Time PCR System
See your instrument user guide for recommended calibration schedules and detailed calibration
instructions.

Plate Cat. No.[1]

96-well 0.2 mL

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well A24738

FAM™ Dye Spectral Calibration Plate, 96-well 4432327

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well A24737

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well 4461599

VIC™ Dye Spectral Calibration Plate, 96-well 4432334

96-well 0.1 mL

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well A24734

FAM™ Dye Spectral Calibration Plate, Fast 96-well 4432389

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well A24735

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well 4457328

VIC™ Dye Spectral Calibration Plate, Fast 96-well 4432396

384-well

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well A24736

FAM™ Dye Spectral Calibration Plate, 384-well 4432271

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well A24733

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well 4457334

VIC™ Dye Spectral Calibration Plate, 384-well 4432278

[1] Catalog numbers that appear as links open the web pages for those products.

TaqMan™ SCID/SMA Plus Assay User Guide 9

 Chapter 2 Calibration information
2 Dye spectral calibration plates for the QuantStudio™ 5 Real‑Time PCR System

Dye spectral calibration plates for the QuantStudio™ 5

Real‑Time PCR System
See your instrument user guide for recommended calibration schedules and detailed calibration
instructions.

Plate Cat. No.[1]

96-well 0.2 mL

QuantStudio™ 3/5 Spectral Calibration Plate 1

A26331
(FAM™, VIC™, ROX™, and SYBR™ dyes), 96‑Well 0.2‑mL

QuantStudio™ 3/5 Spectral Calibration Plate 2, 96‑Well 0.2‑mL

A26332
(ABY™, JUN™, and MUSTANG PURPLE™ dyes)

96-well 0.1 mL

QuantStudio™ 3/5 Spectral Calibration Plate 1,

A26336
(FAM™, VIC™, ROX™, and SYBR™ dyes), 96‑well, 0.1 mL

QuantStudio™ 3/5 Spectral Calibration Plate 2 (ABY™, JUN™, MUSTANG PURPLE™

A26337
dyes), 96‑well Fast (0.1‑mL) Plate

384-well

QuantStudio™ 5 Spectral Calibration Plate 1, (FAM™, VIC™, ROX™

A26334
TAMRA™, and SYBR™ dyes), 384-well

QuantStudio™ 5 Spectral Calibration Plate 2 (ABY™, JUN™, MUSTANG PURPLE™,

A26335
NED™, and Cy®5 dyes), 384-well
[1] Catalog numbers that appear as links open the web pages for those products.

Dye spectral calibration plates for the QuantStudio™ Dx

Real‑Time PCR Instrument (RUO mode)
See your instrument user guide for recommended calibration schedules and detailed calibration
instructions.

Plate Cat. No.[1]

96-well 0.2 mL

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well A24738

FAM™ Dye Spectral Calibration Plate, 96-well 4432327

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well A24737

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, 96-well 4461599

VIC™ Dye Spectral Calibration Plate, 96-well 4432334

10 TaqMan™ SCID/SMA Plus Assay User Guide

 Chapter 2 Calibration information
Dye spectral calibration plates for the QuantStudio™ Dx Real‑Time PCR Instrument (RUO mode) 2

(continued)

Plate Cat. No.[1]

96-well 0.1 mL

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well A24734

FAM™ Dye Spectral Calibration Plate, Fast 96-well 4432389

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well A24735

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, Fast 96-well 4457328

VIC™ Dye Spectral Calibration Plate, Fast 96-well 4432396

384-well

ABY™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well A24736

FAM™ Dye Spectral Calibration Plate, 384-well 4432271

JUN™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well A24733

MUSTANG PURPLE™ Dye Spectral Calibration Plate for Multiplex qPCR, 384-well 4457334

VIC™ Dye Spectral Calibration Plate, 384-well 4432278

[1] Catalog numbers that appear as links open the web pages for those products.

TaqMan™ SCID/SMA Plus Assay User Guide 11

 3 Methods

■ Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
■ Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
■ Prepare the standard curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
■ Extract the DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
■ Prepare the real-time PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
■ Set up and run the real-time PCR instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
■ Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

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 16.
• 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 16.
• Ensure that the instrument is calibrated for each detector dye and passive reference dye, according
to the instrument user guide. See Chapter 2, “Calibration information”.

12 TaqMan™ SCID/SMA Plus Assay User Guide

 Chapter 3 Methods
Before you begin 3

Before you begin

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

• Prepare DBS wash buffer—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 buffer.
DBS wash buffer 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 buffer—add 0.5 mL of 10% Tween®-20 Surfact-Amps™ Detergent Solution to

100 mL of TE Buffer.
Stabilizing buffer is modified TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8) with 0.05% Tween®-20
detergent added.

Note: Discard the Stabilizing Solution provided in the Extract All Kit. This assay workflow utilizes
the prepared stabilizing buffer as described in this section.

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)

TREC 18ACJFZP 2747 bp

KREC 18ACJFTP 2786 bp

1. Re-suspend the lyophilized plasmid with Low TE:

a. For a 5 μg lyophilized plasmid, add 500 μL of Low TE to the tube.

b. Vortex and centrifuge the tube.

c. Let it sit on benchtop for 5-10 minutes at room temperature.

d. Vortex and centrifuge the tube again.

Target Concentration Approximate copies per μL

TREC 10 ng/μL 3.37 x 109 copies/µL
KREC 10 ng/μL 3.33 x 109 copies/µL

TaqMan™ SCID/SMA Plus Assay User Guide 13

 Chapter 3 Methods
3 Extract the DNA

2. Dilute the TREC plasmid by 1:10 in prepared stabilizing buffer (for example, add 5 µL of plasmid
to prepare 50 µL total volume in stabilizing buffer—0.05% Tween®-20 detergent in low TE buffer),
then denature for 10 minutes at 95°C to linearize the plasmid.
The final concentration of the linearized plasmid will be approximately 3.3 x 108 copies/µL.

Note: Linearization of the plasmid is required only once.

3. Dilute the linearized plasmid solution by 1:10 in stabilizing buffer to prepare a stock.
The final concentration of the stock will be approximately 3.3 x 107 copies/µL. The stock can be
stored at 4°C for short-term use.

4. Prepare serial dilutions as needed. Mix each dilution well by vortexing, followed by centrifugation
between each dilution step.
All of the serial dilutions must be diluted in stabilizing buffer and can be stored at 4°C for short-
term use.

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

The recommended standard curve points are as follows.

Approximate copies per

Standard curve Copies per µL of plasmid
reaction[1]

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

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] We 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.

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 buffer to each sample well.
• 1.5 mm DBS punch: 70 µL
• 3.2 mm DBS punch: 150 µL

14 TaqMan™ SCID/SMA Plus Assay User Guide

 Chapter 3 Methods
Extract the DNA 3

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 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.

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 buffer to each sample well.

Note:
· The total lysate volume per 1.5 mm or 3.2 mm DBS punch is 40 µL.
· Use the stabilizing buffer prepared in “Before you begin” on page 13.

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

TaqMan™ SCID/SMA Plus Assay User Guide 15

 Chapter 3 Methods
3 Prepare the real-time PCR reactions

Prepare the real-time PCR reactions

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

Volume per reaction[1]

Component
96-well plate 384-well plate
™ ™
2X TaqPath ProAmp Multiplex Master
10 µL 7.5 µL
Mix
20X TaqMan™ SCID/SMA Plus Assay 1 µL 0.75 µL
Total PCR Reaction Mix volume 11 µL 8.25 µL
[1] Add 10% overage for pipetting loss. Excess PCR Reaction Mix can be stored at 2–8°C for up to 48 hours.

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 either the transferred lysate, TREC/KREC
plasmids (in order to generate standard curve), or negative control sample (for example: nuclease-
free water to serve as No-Template Control or NTC).
• 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.

Set up and run the real-time PCR instrument

See your instrument user guide for detailed instructions to program the thermal cycling conditions or to
run the plate.

Note: The instrument must be configured with the block appropriate for the plate type.

1. Select or create dye detectors, then assign them to each well in the plate layout.

Target Reporter Quencher

™
SMN1 FAM dye MGB-NFQ
TREC JUN™ dye QSY™
KREC ABY™ dye QSY™
RNase P VIC™ dye QSY™

2. Set up the following conditions.

• Cycling mode: Fast
• Passive reference: MUSTANG PURPLE™ dye

16 TaqMan™ SCID/SMA Plus Assay User Guide

 Chapter 3 Methods
Analyze the results 3

3. Set the reaction volume.

• Reaction volume for a 1.5 mm DBS punch in a 96-well plate: 20 µL
• Reaction volume for a 3.2 mm DBS punch in a 96-well plate: 20 µL
• Reaction volume for a 3.2 mm DBS punch in a 384-well plate: 15 µL

4. Set up the following thermal cycling protocol.

Step Temperature Time Cycles

Initial denaturation / Enzyme
95°C 20 seconds 1
activation
Denature 95°C 1 second
40
Anneal / Extend 60°C 20 seconds

5. Load the plate into the real-time PCR instrument.

6. Start the run.

Analyze the results

See your instrument user guide for more detailed information about data analysis.

1. View the amplification plots.

2. Set the baseline and threshold values.

3. Calculate the Ct values on the instrument.

4. (For standard curve experiments) View the standard curve for the following items:
• Slope • Y-intercept
• Amplification efficiency • Ct values
• R2 values • Outliers

Overview of baseline and threshold values

You can use the real-time PCR system software to set the baseline and threshold values for the
amplification plot, either automatically or manually.
• The baseline refers to the initial cycles of PCR in which there is slight change in fluorescence signal.
• The intersection of the threshold with the amplification plot defines the Ct in real-time PCR assays.
The threshold is set above the background signal and within the exponential growth phase of the
amplification curve.

TaqMan™ SCID/SMA Plus Assay User Guide 17

 A Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user
documentation may result in personal injury or damage to the instrument or device. Ensure that
anyone using this product has received instructions in general safety practices for laboratories and
the safety information provided in this document.

· Before using an instrument or device, read and understand the safety information provided in the
user documentation provided by the manufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use
appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain
SDSs, see the “Documentation and Support” section in this document.

18 TaqMan™ SCID/SMA Plus Assay User Guide

 Appendix A Safety
Chemical safety A

Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel
read and practice the general safety guidelines for chemical usage, storage, and waste provided
below. Consult the relevant SDS for specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see
the "Documentation and Support" section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with
sufficient ventilation (for example, fume hood).
· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer
cleanup procedures as recommended in the SDS.
· Handle chemical wastes in a fume hood.
· Ensure use of primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary container.
Both containers must be compatible with the waste material and meet federal, state, and local
requirements for container storage.)
· After emptying a waste container, seal it with the cap provided.
· Characterize (by analysis if needed) the waste generated by the particular applications, reagents,
and substrates used in your laboratory.
· Ensure that the waste is stored, transferred, transported, and disposed of according to all local,
state/provincial, and/or national regulations.
· IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal
limitations may apply.

AVERTISSEMENT ! PRÉCAUTIONS GÉNÉRALES EN CAS DE MANIPULATION DE PRODUITS

CHIMIQUES. Pour minimiser les risques, veiller à ce que le personnel du laboratoire lise attentive‐
ment et mette en œuvre les consignes de sécurité générales relatives à l’utilisation et au stockage
des produits chimiques et à la gestion des déchets qui en découlent, décrites ci-dessous. Consulter
également la FDS appropriée pour connaître les précautions et instructions particulières à respecter :

· Lire et comprendre les fiches de données de sécurité (FDS) fournies par le fabricant avant de
stocker, de manipuler ou d’utiliser les matériaux dangereux ou les produits chimiques. Pour obtenir
les FDS, se reporter à la section « Documentation et support » du présent document.
· Limiter les contacts avec les produits chimiques. Porter des équipements de protection appropriés
lors de la manipulation des produits chimiques (par exemple : lunettes de sûreté, gants ou vête‐
ments de protection).
· Limiter l’inhalation des produits chimiques. Ne pas laisser les récipients de produits chimiques
ouverts. Ils ne doivent être utilisés qu’avec une ventilation adéquate (par exemple, sorbonne).
· Vérifier régulièrement l’absence de fuite ou d’écoulement des produits chimiques. En cas de fuite
ou d’écoulement d’un produit, respecter les directives de nettoyage du fabricant recommandées
dans la FDS.
· Manipuler les déchets chimiques dans une sorbonne.

TaqMan™ SCID/SMA Plus Assay User Guide 19

 Appendix A Safety
A Biological hazard safety

· Veiller à utiliser des récipients à déchets primaire et secondaire. (Le récipient primaire contient les
déchets immédiats, le récipient secondaire contient les fuites et les écoulements du récipient pri‐
maire. Les deux récipients doivent être compatibles avec les matériaux mis au rebut et conformes
aux exigences locales, nationales et communautaires en matière de confinement des récipients.)
· Une fois le récipient à déchets vidé, il doit être refermé hermétiquement avec le couvercle fourni.
· Caractériser (par une analyse si nécessaire) les déchets générés par les applications, les réactifs et
les substrats particuliers utilisés dans le laboratoire.
· Vérifier que les déchets sont convenablement stockés, transférés, transportés et éliminés en res‐
pectant toutes les réglementations locales, nationales et/ou communautaires en vigueur.
· IMPORTANT ! Les matériaux représentant un danger biologique ou radioactif exigent parfois une
manipulation spéciale, et des limitations peuvent s’appliquer à leur élimination.

WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the instrument is

potentially hazardous. Follow the guidelines noted in the preceding General Chemical Handling
warning.

WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and waste bottles can crack
and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the
cover fastened and the handles locked in the upright position.

Biological hazard safety

WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface
may be considered a biohazard. Use appropriate decontamination methods when working with
biohazards.

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents,
and blood of humans and other animals have the potential to transmit infectious diseases.
Conduct all work in properly equipped facilities with the appropriate safety equipment (for example,
physical containment devices). Safety equipment can also include items for personal protection,
such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or
goggles. Individuals should be trained according to applicable regulatory and company/ institution
requirements before working with potentially biohazardous materials. Follow all applicable local,
state/provincial, and/or national regulations. The following references provide general guidelines when
handling biological samples in laboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical
Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009;
found at:
https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-
P.pdf
· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

20 TaqMan™ SCID/SMA Plus Assay User Guide

 B Documentation and support

Related documentation
Document Pub. No.

TaqMan™ SCID/SMA Plus Assay Quick Reference MAN0019380

TaqMan™ SCID/SMA Plus Assay Calibration Guide MAN0019382

Customer and technical support

Visit thermofisher.com/support for the latest service and support information.
• Worldwide contact telephone numbers
• Product support information
– Product FAQs
– Software, patches, and updates
– Training for many applications and instruments
• Order and web support
• Product documentation
– User guides, manuals, and protocols
– Certificates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers, contact the
manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the
Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/
global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at
www.thermofisher.com/support.

TaqMan™ SCID/SMA Plus Assay User Guide 21

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11 August 2021