Instructions for Use

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0

For identification of T-cell clonality.
This assay is for Research Use Only. Not for use in diagnostic procedures.

Manufactured in U.S.A.

Storage Conditions: -85ºC to -65ºC(DNA controls may be separated from assay kits and stored at 2°C to 8°C)

Catalog #12070101
12070111

Products

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 MegaKit

Quantity

33 Reactions
330 Reactions

Page 2 of 3

Table of Contents
1.

ASSAY USE .............................................................................................................................................................................................. 32.

SUMMARY AND EXPLANATION OF THE TEST ...................................................................................................................................................... 3
2.1.
2.2.

3.

Background ....................................................................................................................................................................................................... 3
Summary ........................................................................................................................................................................................................... 3

PRINCIPLES OF THE PROCEDURE .................................................................................................................................................................... 4
3.1.
3.2.
3.3.

4.

Polymerase Chain Reaction (PCR) ..................................................................................................................................................................... 4
Gel Detection .................................................................................................................................................................................................... 4
Differential Fluorescence Detection.................................................................................................................................................................. 4

REAGENTS ............................................................................................................................................................................................... 5
4.1.
4.2.
4.3.

5.

Reagent Components ........................................................................................................................................................................................ 5
Warnings and Precautions ................................................................................................................................................................................ 5
Storage and Handling ........................................................................................................................................................................................ 6

INSTRUMENTS .......................................................................................................................................................................................... 7
5.1.
5.2.
5.3.
5.4.

6.

Thermal cycler ................................................................................................................................................................................................... 7
Electrophoresis Unit (for Gel Detection) ........................................................................................................................................................... 7
UV Illumination Unit (for Gel Detection) ........................................................................................................................................................... 7
ABI Capillary Electrophoresis Instruments ........................................................................................................................................................ 7

SPECIMEN COLLECTION AND PREPARATION ...................................................................................................................................................... 8
6.1.
6.2.
6.3.
6.4.
6.5.
6.6.

7.

Precautions ....................................................................................................................................................................................................... 8Interfering Substances ...................................................................................................................................................................................... 8Specimen Requirements ................................................................................................................................................................................... 8
Sample Preparation........................................................................................................................................................................................... 8
Template Amplification Controls....................................................................................................................................................................... 8
Sample Storage ................................................................................................................................................................................................. 8

ASSAY PROCEDURE .................................................................................................................................................................................... 9
7.1.
7.2.
7.3.
7.4.
7.5.
7.6.

8.

Materials Provided ............................................................................................................................................................................................ 9Reagents Required (not provided) .................................................................................................................................................................... 9Reagent Preparation ....................................................................................................................................................................................... 10
Amplification ................................................................................................................................................................................................... 11
Detection......................................................................................................................................................................................................... 11
Recommended Positive Controls .................................................................................................................................................................... 13

INTERPRETATION AND REPORTING ............................................................................................................................................................... 13
8.1.
8.2.
8.3.

9.

Sample Analysis (Manual) ............................................................................................................................................................................... 13
Sample Interpretation (Manual) ..................................................................................................................................................................... 14
Sample Analysis and Interpretation (Automated) ........................................................................................................................................... 14

LIMITATIONS OF PROCEDURE ...................................................................................................................................................................... 16

10.

EXPECTED RESULTS .................................................................................................................................................................................. 17
10.1.
10.2.
10.3.

Expected Size of Amplified Products ............................................................................................................................................................... 17
Sample Data (Manual Interpretation) ............................................................................................................................................................. 17
Sample Data (Automated Interpretation) ....................................................................................................................................................... 18

11.

TECHNICAL AND CUSTOMER SERVICE ............................................................................................................................................................ 18

12.

REFERENCES .......................................................................................................................................................................................... 1913.

SYMBOLS .............................................................................................................................................................................................. 19

14.

LEGAL NOTICE ........................................................................................................................................................................................ 19
14.1.
14.2.

15.

Warranty and Liability ..................................................................................................................................................................................... 19
Patents and Trademarks ................................................................................................................................................................................. 19

T-CELL RECEPTOR GAMMA GENE REARRANGEMENT ASSAY 2.0: SINGLE PAGE GUIDE .............................................................................................. 20

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 3 of 4

1. Assay Use
This Research Use Only assay identifies T-cell receptor gamma (TCRG) chain gene rearrangements and is useful for theidentification of clonal T-cell populations and evaluation of new research and methods in malignancy studies.

2. Summary and Explanation of the Test
2.1.

Background
Invivoscribe’s T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 represents an improved approach to PCRbased clonality testing of T-cell lymphoproliferative disorders. This assay was optimized using a single amplification
reaction with a single fluorescent dye for detection. Amplified products generated targeting the TCR gamma gene
locus all fall within a single size range to facilitate interpretation.
The human TCR gamma gene locus on chromosome seven (7q14) includes 14 V genes (six of these V genes arefunctional; three (3) open reading frames and five (5) pseudogenes) belonging to four subgroups (Group I, II, III andIV), five (5) J segments and two (2) C genes spread over 200 kilobases. The diversity of this locus has complicated
PCR-based testing. This multiplex PCR assay represents an improvement over existing assays as it can detect the vast
majority of TCR gamma gene rearrangements with a single multiplex master mix. Importantly, this assay includes
primers for all known groups of TCR gamma variable region genes and joining region genes involved inrearrangements in T-cell lymphomas. In addition, all labeled primers are conjugated with the 6FAM fluorophore.

2.2.

Summary
Our single multiplex master mix targets all conserved regions within the variable (V) and the joining (J) region genesthat are described in lymphoid malignancies. This is critical for more comprehensive analysis of samples, as some Tcell lymphoproliferative disorders involve V and J segments that would not be identified with a single Vγ (1-8) andJγ1/2 primer set. Amplification with all primers in a single tube has several additional important advantages overexisting methods. The polyclonal background that results from the combination of all primers in a single tube
produces a more robust and easily interpreted signal with capillary electrophoresis, which aids in the interpretationof small peaks. The average size of the TCRG gene rearrangement PCR product is 190 bp, with a normal distributionof product sizes between 159 and 207 bp. This protocol leads to improved PCR product formation from paraffinembedded samples compared to other protocols that yield products of 260 bp or longer. Positive and negative DNA
controls, as well as a Specimen Control Size Ladder master mix are included. Clonality is indicated if a dominant
amplicon is detected.
This test kit includes the TCRG - 6FAM master mix that targets framework regions within the variable region and thejoining region of the TCR gamma chain locus. The second master mix, the Specimen Control Size Ladder, targets
multiple genes and generates a series of amplicons of 96, 197, 297, 397, and 602 bp that can be used to ensure thatthe quality of input DNA is adequate to yield a valid result.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 4 of 5

3. Principles of the Procedure
3.1.

Polymerase Chain Reaction (PCR)
PCR assays are routinely used for the identification of clonal T-cell populations. This test amplifies the DNA between
primers that target conserved regions within the variable (V) and the joining (J) regions that flank the unique
hypervariable antigen-binding region 3 (CDR3). These conserved regions lie on either side of an area within the V-J
region where programmed genetic rearrangements occur during maturation of all B and T lymphocytes. The antigen
receptor genes that undergo rearrangement are the immunoglobulin heavy chain and light chains in B-cells, and the
T-cell receptor genes in T-cells. Each B- and T-cell has a single productive V-J rearrangement that is unique in both
length and sequence. Therefore, when DNA from a normal or polyclonal population is amplified using DNA primersthat flank the V-J region, a Gaussian distribution (bell-shaped curve) of amplicon products is produced within an
expected size range. This Gaussian distribution reflects the heterogeneous population of V-J rearrangements. In
certain cases, where lymphocyte DNA is not present, no product is detected. DNA from samples containing a clonal
population yield one or two prominent amplified products (amplicons) within the valid size range.

Figure 1:

Simple representation of the organization if the T-cell receptor gamma gene on chromosome 7. Black arrows represent the relative
positions of primers that target the variable region genes and the joining downstream joining region gene segments that are involved
in rearrangements in T-cell lymphomas. The downstream primers are fluorescently labeled through the incorporation of a 6 FAM
fluorophore. The amplicon products generated from these rearrangements are detected by capillary electrophoresis.

Since the antigen receptor genes are polymorphic (consisting of a heterogeneous population of related DNA
sequences), it is difficult to employ a single set of DNA primer sequences to target all of the conserved flanking
regions around the V-J rearrangement. N-region diversity, and somatic mutation further scramble the DNA sequencesin these regions. Therefore, a multiplex master mix, which targets multiple V and J regions (Figure 1), is required todetect the majority of clonal rearrangements. As indicated, clonal rearrangements are identified as one or two
prominent, single-sized products within the background of different-sized amplicon products that form the Gaussian
distribution around a statistically favored, average-sized rearrangement.
3.2.

Gel Detection
Gel electrophoresis, such as agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresis(PAGE), is commonly used to resolve amplicon products based on their size, charge, and conformation. Since DNA isnegatively charged, when an electrical potential (voltage) is applied across the gel containing PCR products, theelectrical field causes the amplicons to migrate through the gel. Smaller DNA fragments are able to easily migrate
through the gel matrix, whereas larger DNA fragments migrate more slowly. This causes a separation of the amplicon
products based on size. Ethidium bromide or other DNA intercalating dyes can then be used to stain and detect theseproducts in the gel.

3.3.

Differential Fluorescence Detection
Differential fluorescence detection is commonly used to resolve the different-sized amplicon products using acapillary electrophoresis instrument. Primers can be conjugated with several different fluorescent dyes
(fluorophores) so that they can produce different emission spectra upon excitation by a laser in the capillaryelectrophoresis instrument. In this manner, different fluorescent dyes can correspond to different targeted regions.
This detection system results in unsurpassed sensitivity, single nucleotide resolution, differential product detection
and relative quantification. In addition, the use of agarose and polyacrylamide gels, as well as the use of carcinogens
such as ethidium bromide, can virtually be eliminated. Further, differential detection allows accurate, reproducible
and objective interpretation of primer-specific products and automatic archiving of data. Inter-assay and intra-assay
reproducibility in size determination using capillary electrophoresis is approximately one to two nucleotides.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 5 of 6

4. Reagents
4.1.

Reagent Components

Table 1. Available Kits

Catalog #Description

Total Reactions

12070101

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0

33 Reactions

12070111

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 MegaKit

330 Reactions

Table 2. Reagent Components

Reagent

Part #

Reagent Components (active ingredients)

Unit
Quantity

12070101# of Units

12070111# of Units

1500 µL

1

101500 µL

1

10

50 µL

1

5

Storage
Temp.

TCRG – 6FAM
Master Mixes

22070091

Template
Amplification Control
Master Mix

20960021

Positive Control
DNA40883320

Negative (Normal)
Control DNA
Note:

4.2.

40920020

Multiple oligonucleotides targeting the Vγ2,3, 4, 5, 8, 9, 10, & 11 and Jγ1/Jγ2, JγP, andJγP1/JγP2 regions of the T-cell receptor
gamma gene in a buffered salt solution.

Specimen Control Size Ladder – 6FAM
Multiple oligonucleotides targetinghousekeeping genes.

5% TCRG Positive Control DNA

50 μg/mL of DNA in 1/10th TE solution

TCRG Negative Control DNA

50 μg/mL of DNA in 1/10th TE solution

or
50 µL

1

5

There are no preservatives used in the manufacture of this kit.

Warnings and Precautions














Use this assay kit as a system; do not substitute other manufacturers’ reagents. Dilution, reducing amplification
reactions or other deviations from this protocol may affect the performance of this test and/or nullify any limited
sublicense that come with the purchase of these kits.
Do not mix or combine reagents from kits with different lot numbers.
Materials are stable until the labeled expiration date when stored and handled as directed. Do not use kitsbeyond their expiration date.
Adherence to the protocol will assure optimal performance and reproducibility. Ensure correct thermal cycler
programs are used, as other programs may provide inaccurate/faulty data such as false-positive and falsenegative results.
Dispose of unused reagents and waste in accordance with country, federal, state and local regulations.
Perform all laboratory procedures with standard personal protective equipment (gloves, laboratory coats andprotective eyewear). Follow good laboratory practices and universal precautions when working with specimens.
Do not pipette by mouth. Do not eat, drink or smoke in laboratory work areas. Wash hands thoroughly after
handling specimens and assay reagents. Handle specimens in approved biological safety containment facilities
and open only in certified biological safety cabinets.
Due to the high analytical sensitivity of this test, use extreme care to avoid any contamination of reagents oramplification mixtures with samples, controls or amplified materials. Use fresh, aerosol-resistant pipette tips
between samples and between dispensing reagents. Closely monitor all reagents for signs of contamination(e.g., negative controls giving positive signals). Discard any reagents suspected of contamination.
To minimize contamination, wear clean gloves when handling samples and reagents and routinely clean work
areas and pipettes prior to setting up PCR.
Follow uni-directional workflow between separate work areas in the PCR laboratory: begin with master mix
preparation, move to specimen preparation, then to amplification and finally to detection. Autoclaving does not
eliminate DNA contamination. Perform pre- and post-PCR steps in separate spaces. Avoid taking paper andother materials from post-PCR into the pre-PCR space.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 6 of 7






Dedicate all pipettes, pipette tips and any equipment used in a particular area to that area of the laboratory.
Decontaminate non-disposable items with 10% bleach and rinse with distilled water two separate times before
returning them to the starting areas.
Use sterile, disposable plastic ware whenever possible to avoid contamination.
The IdentiClone RUO TCRG 2.0 Software is an automated method analysis software for the capillaryelectrophoresis data obtained from ABI 3500 platform.

IMPORTANT:


4.3.

The IdentiClone RUO TCRG 2.0 Software can only be used with the files generated from the ABI 3500
series instruments. Sample analysis using the IdentiClone RUO TCRG 2.0 software on ABI 3500
Platforms requires configuration of a platemap in the software corresponding to the ABI detection plate.
Refer to Software IFU (: 310027) section 6.5. for detailed instructions on plate setup. Following theplate setup in the software, a CSV format sample sheet will be generated for use with the ABI 3500
instrument.

The IdentiClone RUO TCRG 2.0 Software requires inclusion of all three controls in each run - Positive Control ( :40883320), Negative Control ( : 40920020), and No Template Control (nuclease free water or sample diluent).

Storage and HandlingStore the assay at -85ºC to -65ºC until ready to use.
All reagents and controls must be thawed and vortexed or mixed thoroughly prior to use to ensure that they arecompletely resuspended.
Due to high salt concentrations, PCR master mixes are sensitive to freeze/thaw cycles. Minimize the exposure ofmaster mixes to freeze/thaw cycles.

If you have any questions, please contact the Invivoscribe technical staff. We would be happy to help you determine
your optimal storage needs.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 7 of 8

5. Instruments
5.1.

Thermal cycler

Use or function: Amplification of DNA samples
Performance characteristics and specification:
o
o



5.2.

Follow manufacturer’s installation, operation, calibration, and maintenance procedures.
See section 7.4. Amplification for thermal cycler program.

Electrophoresis Unit (for Gel Detection)

Use or function: DNA fragment separation
Performance characteristics and specification:
o


5.3.

Capable of running at 35V to 135V for extended times

Follow manufacturer’s installation, operation, calibration, and maintenance procedures.

UV Illumination Unit (for Gel Detection)

Use or function: DNA detection
Performance characteristics and specification:
o


5.4.

Minimum thermal range: 15ºC to 96ºC
Minimum ramping speed: 0.8ºC/sec

Capable of emitting light at a wavelength of ~302 nm

Follow manufacturer’s installation, operation, calibration, and maintenance procedures.

ABI Capillary Electrophoresis Instruments

Use or function: Fragment detection and analysis
Performance characteristics and specification:o

The following capillary electrophoresis instruments will meet the performance needs for this assay:
•
•
•
•
•
•
•






ABI 310 Avant Genetic Analyzer (4-capillaries)
ABI 3100 Avant Genetic Analyzer (4-capillaries)
ABI 3100 Genetic Analyzer (16-capillaries)
ABI 3130 Genetic Analyzer (4-capillaries)
ABI 3130xL Genetic Analyzer (16-capillaries)
ABI 3500 Genetic Analyzer (8-capillaries)
ABI 3500xL Genetic Analyzer (24-capillaries)

Follow manufacturer’s installation, operation, calibration, and maintenance procedures.
The ABI instrument used must be calibrated with appropriate Matrix Standards as outlined in section 7.2. :Reagents Required (not provided).
Use the default settings for your polymer and capillary type.
See section 7.5. Detection.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 8 of 9

6. Specimen Collection and Preparation
6.1.

Precautions
Biological specimens from humans may contain potentially infectious materials. Handle all specimens in accordancewith your institute’s Bloodborne Pathogen program and/or Biosafety Level 2.

6.2.

Interfering Substances
The following substances are known to interfere with PCR:





6.3.

Divalent cation chelators
Low retention pipette tips
EDTA (not significant at low concentrations)
HeparinSpecimen Requirements
This assay tests extracted and purified genomic DNA (gDNA). Common sources of gDNA include:



6.4.

5 cc of peripheral blood, bone marrow biopsy or bone marrow aspirate anti-coagulated with heparin or EDTA; OR
Formalin-fixed paraffin embedded tissue or slides.

Sample Preparation

6.4.1.
6.4.2.

Test all samples in duplicate.
Include positive, negative and no template controls with each master mix tested.
Using any method of DNA extraction, extract the gDNA from unknown samples.
Resuspend DNA to final concentration of 20 μg – 200 μg per mL in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or
distilled water.



6.5.

This is a robust assay system. A wide range of DNA concentrations will generate a valid result. Therefore,
quantifying and adjusting DNA concentrations is generally not necessary.
When testing unknown samples, it is recommended to use a template amplification control master mix(e.g., Specimen Control Size Ladder) to verify the absence of PCR inhibitors and sufficient quality andquantity of DNA is present to generate a valid result.

Template Amplification Controls
The Specimen Control Size Ladder master mix primers are labeled with a fluorescent dye (6-FAM). This label isdetected as BLUE using the differential fluorescence software. The amplicons produced with this master mix are at~96, 197, 297, 397, and 602 bp. Please note that the ~96 bp band is comprised of 84 bp and 96 bp bands. Both ofthese bands co-migrate on a gel. Run the products of this master mix separately.

6.6.

Sample Storage
Store samples using a method that prevents degradation of DNA.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 9 of 10

7. Assay Procedure
7.1.

Materials Provided
See Table 2 for materials provided.

7.2.

Reagents Required (not provided)

Table 3. Materials Required (not provided)

Reagent/Material

DNA Polymerase

Recommended Reagents/Materials and Suppliers
Roche:• EagleTaq DNA Polymerase
Invivoscribe:• FalconTaq DNA Polymeraseor equivalentPart Number

Notes

05206944190
60970130

N/A

Molecular Biology Gradeor USP Water

N/A

N/A

DNase / RNase free

Calibrated Pipettes

N/A

N/A

Capable of accurately
measuring volumes between
1 μL and 1000 μL.

N/A

N/A

Thermal cycler

Thermo Fisher Scientific:• Veriti Dx Thermal Cycler
Bio-Rad:• MJ Research PTC-100 or PTC-200, PTC-220, PTC-240
Perkin-Elmer• PE 9600 or PE 9700

Vortex Mixer

N/A

N/A

N/A

PCR plates or tubes

N/A

N/A

SterileFilter barrier pipette tips

N/A

N/A

Sterile, RNase/ DNase /Pyrogen-free

Microcentrifuge tubes

N/A

N/A

SterileGel Detection
Thermo Fisher Scientific:• UltraPureTM 10 mg/mL Ethidium Bromide

15585-011

6% Polyacrylamide Gels

Thermo Fisher Scientific:• Novex TBE Gels (6%, 12 well)EC62652Box

TBE Running Buffer

Thermo Fisher Scientific:• Novex TBE Running Buffer (5X)

LC6675

Gel Loading Buffer

Thermo Fisher Scientific:• 10X BlueJuiceTM Gel Loading Buffer• Novex Hi-Density TBE Sample Buffer (5X)

10816-015
LC6678100 bp DNA Ladder

Thermo Fisher Scientific:• TrackItTM 100 bp DNA Ladder

10488-058

Ethidium Bromide

Gel Electrophoresis Unit
Ethidium Bromide

N/A
Thermo Fisher Scientific:• UltraPureTM 10 mg/mL Ethidium Bromide

N/A15585-011

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

N/A
N/A
Dilute 1:5 prior to use.

N/A

N/A
N/A
N/A

280288 Rev. H | January 2024

Page 10 of 11
Table 3. Materials Required (not provided)

Reagent/Material

Recommended Reagents/Materials and SuppliersPart Number

Notes

N/A

N/A

ABI Fluorescence Detection
ABI Capillary
Electrophoresis
Instrument

Thermo Fisher Scientific:• ABI 310, 3100, 3130, or 3500 series

Hi-Di Formamide

Thermo Fisher Scientific:• Hi-DiTM Formamide

Size Standards

Invivoscribe:• Hi-Di Formamide w/ROX size standards for ABI 310• Hi-Di Formamide w/ROX size standards for ABI 3100
Thermo Fisher Scientific:• For ABI 3100 or 3130 instruments:
o GeneScanTM - 400HD [ROX]TM• For ABI 3500 instruments:
o GeneScan - 600 [LIZ]TM v2.0
Thermo Fisher Scientific:• 10X Genetic Analyzer Buffer with EDTA

BufferSpectral Calibration Dye
Sets

Thermo Fisher Scientific:• POP-4TM Polymer:o POP-4 for 310 Genetic Analyzerso POP-4 for 3100/3100-Avant Genetic Analyzerso POP-4 for 3130/3130xL Genetic Analyzers• POP-7TM Polymer:o POP-7 for 3130/3130xL Genetic Analyzerso POP-7 for 3500/3500xL Genetic Analyzers

Polymer

7.3.

Thermo Fisher Scientific:• For ABI 3100 and 3130 instruments:o DS-30 Matrix Standard Kit (Dye Set D)• For ABI 310 instruments:
o NED Matrix Standardo And Fluorescent Amidite Matrix Standards [6FAM,
TET, HEX, TAMRA, ROX]
• For ABI 3500 instruments:o DS-33 Matrix Standard Kit (Dye Set G5)

4311320N/A

60980051
60980061
N/A
4029854408399402824

Dilute 1:10 in sterile water
before use

4345827
402996

N/A4015464345833

40283843163554352755

N/A

43527594393714

Reagent Preparation
7.3.1.
7.3.2.

Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex tomix.
In containment hood or dead air box, aliquot the appropriate volume of each master mix into a clean, sterile
microfuge tube.


7.3.3.

Aliquot volumes are 45 μL for each sample + 135 μL (3 x 45 µL) for the positive, negative and no templatecontrols.
Add an additional 20 μL to correct for pipetting errors.

Add the appropriate amount of Taq DNA polymerase (0.25 μL @5 U/μL per 50 μL total reaction volume) toeach master mix and gently mix by inverting several times or gently vortexing.
The master mixes are now ready for distribution to reaction tubes or plate and addition of sample.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 11 of 12

7.4.

Amplification
7.4.1.
7.4.2.
7.4.3.

Aliquot 45 μL of the master mix/enzyme solutions into individual PCR wells or tubes.
Add 5 μL of sample or control DNA to the individual tubes or wells containing the respective master mix
reactions. Pipette up and down several times to mix.
Amplify the reactions using the PCR program in Table 4:

Use the calculated option for temperature measurement with the PTC instruments.
Table 4. Thermal cycling conditions

Standard Program

7.4.4.
7.5.

Step

Temperature

Duration

Cycles1

95ᐤC

7 minutes

1

2

95ᐤC

45 seconds

3

60ᐤC

45 seconds

4

72ᐤC

90 seconds

5

72ᐤC

10 minutes

1

6

15ᐤC

∞

1

35

Remove the amplified PCR plate from the thermal cycler.

Detection
7.5.1.

Gel Detection – Heteroduplex Analysis

7.5.1.1.
7.5.1.2.
7.5.1.3.
7.5.1.4.
7.5.1.5.
7.5.1.6.

Denature 20 μL of PCR products at 94ºC for 5 minutes.
Re-anneal PCR products at 4ºC for 60 minutes.
Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel (made with 1X TBE)
and 0.5X TBE running buffer.
Add 5 μL of ice-cold non-denaturing bromophenol blue loading buffer to samples.
Load 20 μL of mixture into wells of the gel.
Run gel at 110V for 2-3 hours or 40-50V overnight.


7.5.1.7.
7.5.1.8.
7.5.1.9.
7.5.1.10.
7.5.2.

Stain the gels in 0.5 μg/mL Ethidium Bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.
Destain the gels 2X in water for 5-10 minutes.
Visualize the gel using UV illumination.
Photograph the gel and interpret the data.

ABI Fluorescence Detection with ABI 310, 3100 & 3130 instruments

7.5.2.1.
7.5.2.2.
7.5.2.3.
7.5.2.4.
7.5.2.5.
7.5.2.6.
7.5.2.7.

In a new microcentrifuge tube, mix an appropriate amount (10 µL per reaction) of Hi-Di Formamide withROX Size Standards.a Vortex well.
In a new 96-well PCR plate, add 10 µL of Hi-Di Formamide with ROX size standards to individual wells foreach reaction.
Transfer 1 µL of each reaction to the wells containing Hi-Di Formamide with ROX size standards. Add
only one sample per well. Pipette up and down to mix.
Cap or cover the PCR plate.
Heat denature the samples at 95ºC for 2 minutes, then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list for the samples.
Run the samples on an ABI 310/3100/3130 capillary electrophoresis instrument according to the user
manual.


7.5.3.

Voltage and electrophoresis time depend on the PCR amplicon size, acrylamide gel thickness andtype of PCR equipment.
Voltage and run time can be adapted accordingly.

Data are automatically displayed as size and color specific peaks. Review profile and controls,
report results.

ABI Fluorescence Detection with ABI 3500 series instruments

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 12 of 13

Note:

Due to ABI 3500 platform performance variability instrument to instrument, the amount of formamide, sample
and size standard listed in the protocol serves as initial guidelines. Optimization of the protocol may be required
for specific ABI 3500 Platforms.

Important:IdentiClone RUO TCRG 2.0 Software requires configuration of plate map for setting up the detection
plate. Refer to Software Instructions for Use (IFU) (: 310027), section 6.4, for detailed
instructions. Software analysis requires inclusion of all three controls in each run, starting from PCR
through detection.


7.5.3.1.
7.5.3.2.
7.5.3.3.

In a new microcentrifuge tube, mix an appropriate amount (9.5 μL per reaction) of Hi-Di Formamide
with LIZ Size Standards. Vortex well.
In a new 96-well PCR plate, add 9.5 μL of Hi-Di Formamide with LIZ size standards to individual wells foreach reaction.
Transfer 0.5 μL of each reaction to the wells containing Hi-Di Formamide with LIZ size standards.


7.5.3.4.
7.5.3.5.
7.5.3.6.




Note:

If using IdentiClone RUO TCRG 2.0 Software, follow section 6.5. in the software IFU (: 310027)
to create a plate map.
If using IdentiClone RUO TCRG 2.0 Software, enter the ABI 3500 instrument settings listed in Section
8.3. of this IFU.

Run the samples on an ABI 3500 capillary electrophoresis instrument according to the user manual.

a

Add only one sample per well.
Pipette up and down to mix.

Cap or cover the PCR plate.
Heat denature the samples at 95ºC for 3 minutes, then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list to track sample information.



7.5.3.7.

Positive Control ( : 40883320)
Negative Control ( : 40920020)
No Template Control (Water or Sample diluent)

Data are automatically displayed as size and color specific peaks. Review profile and controls,
report results.
If using the IdentiClone RUO TCRG 2.0 Software for data analysis and interpretation, refer to section
7 in the IdentiClone RUO TCRG 2.0 Software IFU (: 310027) for detailed instructions.

Please see Applied Biosystems’ accompanying product insert for mixing Hi-Di Formamide with size standards fordifferent ABI instruments.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

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7.6.

Recommended Positive Controls
The amplicon sizes listed in Table 5 were determined using an ABI platform. The amplicon sizes measured on eachspecific capillary electrophoresis instrument may differ by 1 to 4 base pairs from those listed depending on theplatform of detection and the version of the analysis software used. Once identified, the amplicon size as
determined on a specific platform will be consistent from run to run.
Note:

“Color” indicates the color of products generated with the master mix when using the default color assignment
on ABI fluorescence detection systems.

Table 5. Positive ControlsaMaster Mix

TargetColorControl DNA

Catalog #Product Size (bp)

TCRG - 6FAM

Vγ1-Vγ11+ Jγ1/Jγ2, JγP,JγP1/JγP2

BlueSpecified Size Range
5% TCRG Positive Control DNA

40883320

159-207
194, 196

Specimen Control
Size LadderMultiple GenesBlueTCRG Negative Control DNA

40920020

84, 96, 197, 297, 397, 602a:

Note:

Because smaller PCR fragments are preferentially amplified, it is not unusual for the 602 bp fragment to have a diminished signal or to be
missing entirely. For ABI fluorescence detection, the 602 bp peak may not appear during normal run times. In addition, the size of this peak
may differ by over 30 bp when fragment size is extrapolated using the GeneScan - 400HD [ROX] size standards

8. Interpretation and Reporting
This assay is for research use only; not intended for diagnostic purposes. PCR based testing does not identify 100% of clonalcell populations; therefore, repeat testing by NGS may be advisable to rule out clonality.
8.1.

Sample Analysis (Manual)
8.1.1.
8.1.2.
8.1.3.

Results can be reported as “Clonal”, “Non-Clonal” or “Invalid” for “Detection of T-cell receptor gamma chain
gene rearrangement consistent with the presence of a clonal cell population”.
Report samples that fail to amplify following repeat testing as “A result cannot be reported on this specimen
because there was DNA of insufficient quantity or quality for analysis”.
All assay controls must be examined prior to interpretation of sample results. If the controls do not yield thecorrect results, the assay is not valid and the samples cannot be interpreted.
The following describes the analysis for each control and the decisions necessary based upon the results:

Negative Control: (Polyclonal control, water or no template blank). If the negative control is:oPositive:o

Negative:

Positive Control:oo8.1.4.

Possible contamination of all PCR amplification reactions. Do not continue with theinterpretation of results. Prepare fresh master mix and repeat amplification.
Continue with the analysis.

Positive:
Negative:(This can also be an extraction control if positive control material is taken throughextraction processes). If the positive control is:Continue with analysis.
Repeat assay.

Specimen Control Size Ladder: (This is run on unknown samples only). If the Specimen Control Size Ladder:Generates ~96, 197, 297, 397, and 602 bp products: Please note smaller PCR fragments are
preferentially amplified, and it is not unusual for the 602 bp fragment to havea diminished signal or to be missing entirely. Continue with analysis.
Generates no peaks:
Repeat the assay and/or obtain a new specimen.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 14 of 15

8.2.

Sample Interpretation (Manual)
Following the acceptance of the controls, the samples are interpreted as follows:
8.2.1.

Criteria for defining a positive peak are as follows:
Products generated from samples that fall within the 159 bp - 207 bp size range and, if using differential
fluorescence detection, are at least three times the amplitude of either adjacent peak that might bepresent in the polyclonal background are consistent with a positive peak.
One or two prominent peaks within the specified size range (159 bp - 207 bp) are reported as: “Detectionof T-cell receptor gamma gene rearrangement(s) consistent with the presence of a clonal cell
population.”





Note:

8.3.

The size of amplified products can vary from instrument to instrument by 1 bp - 4 bp. In addition, valid
clonal TCR gamma rearrangements can occur outside the specified size range. Product(s) that are suspect
TCR gamma gene rearrangement(s) that lie outside the specified size range can be sequenced to confirm
their identity.

Sample Analysis and Interpretation (Automated)
IMPORTANT:

Follow the ABI 3500 platform setup, following the instructions below:
8.3.1.

Automated ABI 3500 Platform Setup

8.3.1.1.
8.3.1.2.
8.3.1.3.
8.3.1.4.
8.3.1.5.
8.3.1.6.
8.3.1.7.
8.3.1.8.
8.3.1.9.
8.3.1.10.
8.3.2.

Automated setup can be completed following section 8.3.1, which provides instructions to import
the CSV files included with the IdentiClone RUO TCRG 2.0 Software package.
Manual setup instructions can be completed following section 8.3.2.

Navigate to the XML files included with the software package installed from the IdentiClone RUO TCRG
2.0 Software package.
Follow manufacturer’s instructions to navigate to the Assays library on the ABI instrument.
Click Import and select the TCRG Instrument Parameters file.
Verify the parameters imported on the instrument match the parameters included in Table 6(Instrument Protocol) and Table 7 (Sizecalling Parameters) and click Save.
Follow manufacturer’s instructions to navigate to the File Name Convention library on the ABI
instrument.
Click Import and select the IC_RUO_TCRG_FNC file.
Verify the parameters imported on the instrument match the parameters included in Table 8 and click
Save.
Follow manufacturer’s instructions to navigate to the Results Group library on the ABI instrument.
Click Import and select the IC_RUO_TCRG_RG file.
Verify the parameters imported on the instrument match the parameters included in Table 9 and click
Save.

Manual ABI 3500 Platform Setup

8.3.2.1.
8.3.2.2.
8.3.2.3.
8.3.2.4.
8.3.2.5.

Follow manufacturer’s instructions to navigate to the Assays library on the ABI instrument.
Create a new assay name: TCRG Instrument Parameters.
Select Fragment under Application Type.
Create a new Instrument Protocol under the assay TCRG Instrument Parameters, following theparameters included in Table 6, then click Apply to Assay.
Create new SizeCalling Parameters under the assay TCRG Instrument Parameters, following theparameters included in Table 7, then click Apply to Assay.
Table 6. Instrument Protocol Parameters

Parameter

Value

Protocol NameIC_RUO_TCRG_IP

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

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Table 6. Instrument Protocol Parameters

Parameter

Value

Run Module

Fragment Analysis Module(name could vary)

Application Type

Fragment

Capillary Length

50 cm

Polymer

POP-7

Dye SetG5Disable Name Filter

Unchecked

Oven Temperature

60°C

Run Voltage

19.5 kVolts

PreRun Voltage

15 kVolts

Injection Voltage

1 kVolts

Run Time

1630 seconds

PreRun Time180 seconds

Injection Time

7 seconds

Data Delay

1 second
Advanced Options

Voltage Tolerance

0.7 kVoltsFirst Read Out Time

200 msNormalization Target

4500.0

Voltage # of Steps

30 nk

Second Read Out Time

200 msNormalization Factor Threshold Min

0.3

Voltage Step Interval

15 seconds

Normalization Factor Threshold Max

3.0

Table 7. SizeCalling Parameters

Parameter

Value

Protocol NameIC_RUO_TCRG_SC

Size Standard

GS600LIZ

Sizecaller

SizeCaller v1.1.0

Analysis Range

Full

Sizing Range

Full

Size Calling Method

Local Southern

Primer Peak

Present

Minimum Peak Height – Blue

50

Minimum Peak Height – Green50

Minimum Peak Height – Yellow

50

Minimum Peak Height – Red

50

Minimum Peak Height – Purple

50

Minimum Peak Height – Orange50

Use Smoothing

None

Use Baselining (Baseline Window (Pts))

Checked; 51

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 16 of 17
Table 7. SizeCalling ParametersNote:8.3.2.6.
8.3.2.7.

Parameter

Value

Minimum Peak Half Width

2

Peak Window Size

15

Polynomial Degree

5

Slope Threshold Peak Start

0.0

Slope Threshold Peak End

0.0

Verify all parameters and Save the Assay.

Following manufacturer’s instructions to navigate to the File Name Convention library on the ABI
instrument.
Create a new File Name Convention with the conditions included in Table 8, then click Save after
verifying parameters.
Table 8. File Name Convention

8.3.2.8.
8.3.2.9.

Parameter

Value

Name

IC_RUO_TCRG_FNC

Selected Attribute #1

Sample Name

Selected Attribute #2

Underscore (_)

Selected Attribute #3

Well Position

Selected Attribute #4

Underscore (_)

Selected Attribute #5

Date of Run

Selected Attribute #6

Underscore (_)

Selected Attribute #7

Time of Run

Following manufacturer’s instructions, navigate to the Results Group library on the ABI instrument.
Create a new Results Group with the conditions included in Table 9, then Save after verifying
parameters.

Table 9. Results Group

Parameter

Value

Name

IC_RUO_TCRG_RG

Selected Attribute #1

Plate Name

Selected Attribute #2

Underscore (_)

Selected Attribute #3

Assay Name

Selected Attribute #4

Underscore (_)

Selected Attribute #5

Results Group Name

Include an Instrument Run Name Folder

CheckedInclude a Results Group Name Folder

CheckedInclude an Injection Folder

Checked9. Limitations of Procedure
The IdentiClone RUO TCRG 2.0 Assay is subject to interference by degradation of DNA or inhibition of PCR due to heparin orother agents.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 17 of 18

10. Expected Results
10.1.

Expected Size of Amplified Products

Table 10. Expected Size of Amplified Products

Master Mix

TCRG – 6FAM

Specimen Control
Size Ladder
Note:*Note:

10.2.

TargetColorControl DNA

All V and J genes
Vγ9 + Jγ1/Jγ2Vγ10 + Jγ1/Jγ2

Blue
Blue
Blue

Specified Size Range
TCRG Negative Control DNA
5% TCRG Positive Control DNA
5% TCRG Positive Control DNA

Multiple GenesBlue

Any Human DNA

Catalog # (--40920020
40883320
40883320---)

Product Size (bp)159 - 207
159 - 207
194
196
84*, 96, 197, 297, 397, 602

The amplicon sizes listed above were determined using an ABI platform. Amplicon sizes seen on each specific CE instrument may differ 1 bp - 4 bp
from those listed above depending on the platform of detection (ABI) and the version of the analysis software used. Once identified, the ampliconsize as determined on the specific platform used will be consistent from run to run.
An 84 bp peak may be detected and is likely the result of primer-primer interaction.

Sample Data (Manual Interpretation)
The data shown below (Figure 2) was generated using the TCRG - 6FAM Master Mix, then run on an ABI Instrument.

Figure 2. PCR products
generated with the TCRG –6FAM Master Mix were run on
an ABI instrument.

Figure 3. PCR products generated
with the TCRG – 6FAM
Master Mix were
heteroduplexed and run ona polyacrylamide gel.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 18 of 19

10.3.

Sample Data (Automated Interpretation)
Figure 4. A Sample Report generated
by the IdentiClone RUOTCRG 2.0 Software.

11. Technical and Customer Service
Thank you for purchasing our T-Cell Receptor Gamma Gene Rearrangement Assay 2.0. We appreciate your business. We
are happy to assist you in the validation of this assay and will provide ongoing technical assistance to keep the assays
performing efficiently in your laboratory. Technical assistance is most rapidly obtained using our Internet site:
http://www.invivoscribe.com or by sending an email inquiry to: support@invivoscribe.com.
Contact Information
Invivoscribe, Inc
10222 Barnes Canyon Road | Building 1 | San Diego | California 92121-2711 | USA
Phone: +1 858 224-6600 | Fax: +1 858 224-6601 | Business Hours: 7:00AM - 5:00 PM PST/PDT
Technical Service: support@invivoscribe.com | Customer Service: sales@invivoscribe.com | Website: www.invivoscribe.com

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 19 of 20

12. References
1.
2.
3.
4.
5.

Greiner, TC, Rubocki, RJ. Effectiveness of Capillary Electrophoresis Using Fluorescent-Labeled Primers in Detecting TCell Receptor γ Gene Rearrangements. JMD. 2002, 4(3):137-143.
Lawnickie, LC, Rubocki, RJ, Chan, WC, Lytle, DM, Greiner, TC. The Distribution of Gene Segments in T-Cell Receptor γ
Gene Rearrangements Demonstrates the Need for Multiple Primer Sets. JMD. 2003, 5(2):82-87.
Patel, KP, Pan, Q, Wang, Y, Maitta, RW, Du, J, Xue, X, Lin, J, Ratech, H. Comparison of BIOMED-2 Versus LaboratoryDeveloped Polymerase Chain Reaction Assays for Detecting T-Cell Receptor-γ Gene Rearrangements. JMD.
2010, 12(2):226-238.
Sandberg, Y, Verhaaf, B, van Gastel-Mol, EJ, Wolvers-Tettero, ILM, de Vos, J, MacLeod, RAF, Noordzij, JG, Dik, WA, vanDongen, JJM, Langerak, AW. Human T-cell Lines With Well-Defined T-cell Receptor Gene Rearrangements As
Controls For the BIOMED-2 Multiplex Polymerase Chain Reaction Tubes. Leukemia. 2007, 21:230-237.
Armand, M; Derrieux, C; Beldjord, K; Wabeke, T; Lenze, D; Boone, E; Bruggemann, M; Evans, PAS; Gameiro, P; Hummel,
M; Villarese, P; Groenen, PJTA; Langerak, AW; Macintyre, EA; Davi, F. A New and Simple TRG Multiplex PCR
Assay for Assessment of T-cell Clonality: A Comparative Study from the EuroClonality Consortium.
HemaSphere. 3(3):p e255, June 2019.

13. Symbols
The following symbols are used in labeling for Invivoscribe products.
Catalog Number

Expiration Date

Reagent Volume

Manufacturer

Lot Number

Consult Instructions for Use

Storage Conditions

Research Use Only

14. Legal Notice
14.1.

Warranty and Liability

Invivoscribe, Inc. (Invivoscribe®) is committed to providing the highest quality products. Invivoscribe® warrants that the products meet or
exceed the performance standards described in the Instructions For Use, as to products with such an insert. If a product is covered by productspecifications and does not perform as specified, our policy is to replace the product or credit the full purchase price. No other warranties ofany kind, expressed or implied, are provided by Invivoscribe®. Invivoscribe® liability shall not exceed the purchase price of the product.
Invivoscribe® shall have no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability touse its products; product efficacy under purchaser-controlled conditions in purchaser’s laboratory must be established and continuallymonitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blank controls
every time a sample is tested. Ordering, acceptance, and use of product constitutes purchaser acceptance of sole responsibility for assuring
product efficacy and purchaser agreement to the limitation of liability set forth in this paragraph.
This product is for Research Use Only; not for use in diagnostic procedures.

14.2.

Patents and Trademarks

This product is covered by one or more of the following: European Patent Number 1549764, European Patent Number 2418287, European
Patent Number 2460889, Japanese Patent Number 4708029, United States Patent 8859748, and related pending and future applications. Allof these patents and applications are licensed exclusively to Invivoscribe®. Additional patents licensed to Invivoscribe covering some of theseproducts apply elsewhere. Many of these products require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No
license under these patents to use amplification processes or enzymes is conveyed expressly or by implication to the purchaser by the purchaseof this product.
©2024 Invivoscribe, Inc. All rights reserved. The trademarks mentioned herein are the property of Invivoscribe, Inc. and/or its affiliates or(as to the trademarks of others used herein) their respective owners.
T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024

Page 20 of 20

15. T-Cell Receptor Gamma Gene Rearrangement Assay 2.0: Single Page Guide
15.1.
15.2.
15.3.
15.4.
15.5.
15.6.
15.7.
15.8.

Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube for each master mix).
Aliquot volumes are 45 μL for each sample + 135 μL for the positive, negative and no template controls.
Add an additional 20 μL to correct for pipetting errors.
Add the appropriate amount of Taq DNA polymerase (0.25 μL @5 U/μL per 50 μL total reaction volume) to each master mix and gently
mix by inverting several times or gently vortexing.
Aliquot 45 μL of master mix to individual wells of a PCR plate.
Add 5 μL of DNA from the unknown and control samples to individual tubes or wells containing the respective master mix reactions andpipette up and down several times to mix.
Amplify target DNA using the universal thermal cycler program.

Gel Detection – Heteroduplex Analysis

15.9.
15.10.
15.11.
15.12.
15.13.
15.14.

Denature 20 μL of PCR products at 94ºC for 5 minutes.
Re-anneal PCR products at 4ºC for 60 minutes.
Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel (made with 1X TBE) and 0.5X TBE running buffer.
Add 5 μL of ice-cold non-denaturing bromophenol blue loading buffer to samples.
Load 20 μL of mixture into wells of the gel.
Run gel at 110V for 2-3 hours or 40-50V overnight.


15.15.
15.16.
15.17.
15.18.

Voltage and electrophoresis time depend on the PCR amplicon size, acrylamide gel thickness and type of PCR
equipment.
Voltage and run time can be adapted accordingly.

Stain the gels in 0.5 μg/mL Ethidium Bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.
Destain the gels 2X in water for 5-10 minutes.
Visualize the gel using UV illumination.
Photograph the gel and interpret the data.

ABI Fluorescence Detection with ABI 310, 3100 & 3130 instruments

15.19.
15.20.
15.21.

In a new microcentrifuge tube, mix an appropriate amount (10 μL per reaction) of Hi-Di Formamide with ROX Size Standards. Vortex well.
In a new 96-well PCR plate, add 10 μL of Hi-Di Formamide with ROX size standards to individual wells for each reaction.
Transfer 1 μL of each reaction to the wells containing Hi-Di Formamide with ROX size standards.


15.22.
15.23.
15.24.
15.25.

Cap or cover the PCR plate.
Heat denature the samples at 95ºC for 2 minutes, then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list for the samples.
Run the samples on an ABI 310/3100/3130 capillary electrophoresis instrument according to its user manual.


15.26.

Add only one sample per well.
Pipette up and down to mix.

Data are automatically displayed as size and color specific peaks.

Review profile and controls, report results.

ABI Fluorescence Detection with ABI 3500 instruments

15.27.
15.28.
15.29.

In a new microcentrifuge tube, mix an appropriate amount (9.5 μL per reaction) of Hi-Di Formamide with LIZ Size Standards. Vortex well.
In a new 96-well PCR plate, add 9.5 μL of Hi-Di Formamide with LIZ size standards to individual wells for each reaction.
Transfer 0.5 μL of each reaction to the wells containing Hi-Di Formamide with LIZ size standards.
Add only one sample per well.
Pipette up and down to mix.

15.30.
15.31.
15.32.
15.33.

Cap or cover the PCR plate.
Heat denature the samples at 95ºC for 3 minutes, then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list for the samples.
Run the samples on an ABI 3500 capillary electrophoresis instrument according to the user manual.
Data are automatically displayed as size and color specific peaks.

15.34.

Review profile and controls, report results.

T-Cell Receptor Gamma Gene Rearrangement Assay 2.0
For RESEARCH USE ONLY; not for use in diagnostic procedures

280288 Rev. H | January 2024