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Instructions for Use

T Cell Receptor Gamma Gene Gene Rearrangement Assay 2.0

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

Catalog # Products Quantity

1-207-0101 TCRG Gene Rearrangement Assay 2.0 for ABI Fluorescence Detection 33 Reactions

1-207-0111 TCRG Gene Rearrangement Assay 2.0 MegaKit for ABI Fluorescence Detection 330 Reactions

For Identification of T Cell Clonality

For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Invivoscribe Technologies, Inc.

10222 Barnes Canyon Road, Bldg.1

San Diego, CA 92121-2711 USA

invivoscribe.com | [email protected]

Tel 858.224.6600 | Fax 858.224.6601

Manufactured in U.S.A.

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T Cell Receptor Gamma Gene Rearrangement Assay 2.0 280288 Rev. D | March 2018

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Table of Contents

1. NOTICE .............................................................................................................................................................................. 3

2. PRINCIPLE ........................................................................................................................................................................ 3

3. ASSAY USES ...................................................................................................................................................................... 4

4. SPECIMEN REQUIREMENTS ....................................................................................................................................... 4

5. KIT CONTENTS ................................................................................................................................................................ 4

STATEMENT OF WARNINGS ............................................................................................................................................... 5

6. STORAGE CONDITIONS ................................................................................................................................................ 5

7. REAGENTS REQUIRED BUT NOT INCLUDED ......................................................................................................... 5

PCR AMPLIFICATION ................................................................................................................................................................. 5 ABI FLUORESCENCE DETECTION ............................................................................................................................................... 5

8. RECOMMENDED POSITIVE CONTROLS .................................................................................................................. 5

9. PROCEDURE NOTES ...................................................................................................................................................... 6

10. REAGENT PREPARATION ............................................................................................................................................ 6

11. SAMPLE PREPARATION ............................................................................................................................................... 6

12. AMPLIFICATION ............................................................................................................................................................. 7

13. DETECTION ...................................................................................................................................................................... 7

AVAILABLE TEMPLATE AMPLIFICATION CONTROLS .................................................................................................................. 7 ABI FLUORESCENCE DETECTION WITH ABI 310, 3100 & 3130 INSTRUMENTS .......................................................................... 7 ABI FLUORESCENCE DETECTION WITH ABI 3500 INSTRUMENTS............................................................................................... 8

14. INTERPRETATION AND REPORTING ....................................................................................................................... 8

EXPECTED SIZE OF AMPLIFIED PRODUCTS ................................................................................................................................. 8 SAMPLE INTERPRETATION .......................................................................................................................................................... 9

15. LIMITATIONS OF PROCEDURE .................................................................................................................................. 9

16. REFERENCES ................................................................................................................................................................. 10

17. APPENDIX ....................................................................................................................................................................... 10

REAGENTS AND SPECIAL SUPPLIES .......................................................................................................................................... 10 Differential Fluorescence Detection ........................................................................................................................................ 10

18. TROUBLE SHOOTING GUIDE .................................................................................................................................... 10

20. SAMPLE DATA ............................................................................................................................................................... 11

21. TCRG GENE REARRANGEMENT ASSAY 2.0: SINGLE PAGE FLOW CHART ................................................. 12

ABI FLUORESCENCE DETECTION WITH ABI 310, 3100 & 3130 INSTRUMENTS ........................................................................ 12 ABI FLUORESCENCE DETECTION WITH ABI 3500 INSTRUMENTS............................................................................................. 12

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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:

[email protected]. Alternatively, you can call for technical assistance and for information on our

testing kits at (858) 224-6600 between the hours of 8:00 AM and 5:00 PM Pacific Time.

1. Notice

This product is covered by one or more of the following patents and patent applications owned by or

exclusively licensed to Invivoscribe Technologies, Inc. (IVS):, United States Patent No. 7,785,783, United

States Patent Application Number 10/531,106, European Patent Number EP 1549764B1 and other pending

patent applications originating from European Patent Application Numbers 03756746.8 and 047326551.9 (16

countries), Japanese Patent Number JP04708029B2, Japanese Patent Application Number 2006-529437, Brazil

Patent Application Number PI0410283.5, Canadian Patent Application Number 2525122, Indian Patent

Application Number 5792/DELNP/2005, Mexican Patent Application Number PA/a/2005/012102, Chinese

Patent Application Number 200480016603.5 and Korean Patent Application Number 10-2005-7021561.

Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR).

Any necessary license to practice amplification methods or to use amplification enzymes or equipment covered

by third party patents is the responsibility of the user and no such license is granted by Invivoscribe

Technologies, Inc., expressly or by implication. This product is sold FOR RESEARCH USE ONLY; not for

use in diagnostic procedures.

© 2018 Invivoscribe Technologies, Inc. All rights reserved. The trademarks mentioned herein are the property

of Invivoscribe Technologies, Inc. and/or its affiliates or (as to the trademarks of others used herein) their

respective owners.

2. Principle

NOTICE: Invivoscribe’s T Cell Receptor Gamma Gene Rearrangement Assay 2.0 represents an improved

approach to PCR-based 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.

BACKGROUND:

The human TCR gamma gene locus on chromosome 7 (7q14) includes 14 V genes (6 of these V genes are

functional; 3 open reading frames and 5 pseudogenes) belonging to 4 subgroups (Group I, II, III and IV), 5 J

segments and 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

in rearrangements in T-cell lymphomas. In addition, all labeled primers are conjugated with the 6FAM

fluorophore. The assay provides rapid TCRG clonality assessment in 4-6 hours.

Our single multiplex master mix targets all conserved regions within the variable (V) and the joining (J) region

genes that are described in lymphoid malignancies. This is critical for more comprehensive analysis of patient

samples, as some T-cell lymphoproliferative disorders involve V and J segments that would not be identified

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with a single Vg (1-8) and Jg1/2 primer set. Amplification with all primers in a single tube has several

additional important advantages over existing 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 interpretation of small peaks. Competitive amplification of all TCRG gene

rearrangements allows for identification of a quantitative threshold for a positive result and helps to avoid false

positive results. The average size of the TCRG gene rearrangement PCR amplicons is 190 nucleotides, with a

normal distribution of product sizes between 159 and 207 nucleotides. This protocol should lead to improved

product formation from paraffin-embedded samples compared to other protocols that yield products of 260

nucleotides 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.

Capillary electrophoresis provides a powerful method for the detection of amplicons that have been labeled

with primers conjugated with fluorescent dyes. Reaction products are fractionated and detected simultaneously

using the capillary electrophoresis. This detection system results in unsurpassed sensitivity and product

resolution.

This test kit includes the TCRG - 6FAM Master Mix that targets framework regions within the variable region

and the joining 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 100, 200, 300, 400 and 600 base pairs to

ensure that the quality of input DNA is adequate to yield a valid result. This robust Invivoscribe assay can be

used to test DNA extracted from virtually any source.

3. Assay Uses

T Cell Receptor Gamma Chain Gene Rearrangement Assays are useful for the study of:

Identifying clonal T-cell populations highly suggestive of T-cell malignancies

Monitoring and evaluation of disease recurrence

Evaluation of new research and methods in malignancy studies

4. Specimen Requirements

This assay tests genomic DNA

1. 5 cc of peripheral blood, bone marrow biopsy or bone marrow aspirate anti-coagulated with heparin or

EDTA

2. Minimum 5 mm cube of tissue

3. 100 ng of genomic DNA

4. Formalin-fixed paraffin embedded tissue or slides

5. Kit Contents

Controls and Standards IVS Catalog # Concentration

5% TCRG Positive Control DNA

TCRG Negative Control DNA

4-088-3320

4-092-0020 50 μL @ 50 g/ml

50 μL @ 50 g/ml

Master Mixes IVS Catalog # Target

TCRG - 6FAM

Specimen Control Size Ladder

2-207-0091

2-096-0021

Vg1 – Vg11

Multiple Genes

Note: MegaKits contain 10 units of each master mix and 5 units of each Controls and Standards

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STATEMENT OF WARNINGS

The assay kit has been optimized to be used as a system. Do not substitute other manufacturer’s reagents. Dilution,

reducing amplification reaction volumes or other deviation in this protocol may affect the performance of this test and/or

nullify any limited sublicense that comes with the purchase of this testing kit. Close adherence to the protocol will assure

optimal performance and reproducibility. It is recommended that glass distilled de-ionized molecular biology grade water

be used with the preparation of specimen DNA. This can be purchased from several manufacturers. In addition, laboratory

personnel are reminded to wear appropriate personal protective equipment and follow good laboratory practices and

universal precautions when working with specimens. Specimens should be handled in approved biological safety

containment facilities and opened only in certified biological safety cabinets. Please see Section 9 for further details.

6. Storage Conditions

PCR master mixes are sensitive to freeze/thaw cycles. Therefore, for any duration other than immediate use,

our master mixes and assay kits should be stored at -65C to -85C.

The reason for this is quite straightforward: Due to the high salt concentrations in our master mixes, the

effective freezing and thawing temperature of the master mixes is approximately –10C. The temperature in a

standard laboratory –20C freezer can easily reach –10C or warmer during the day when these freezers are

opened on a regular basis. At these temperatures, PCR master mixes may go through multiple freeze/thaw

cycles, resulting in precipitation of the primers. Accordingly, to minimize the exposure of your master mixes to

freeze/thaw cycles, IVS recommends that master mixes be stored at -65C to -85C.

Please note that our DNA standards are best stored at 2C to 8C. However, these standards can be stored at

any lower temperature as long as they are vortexed after thawing and before use to ensure that they are re-

suspended completely.

If you have any questions, please contact our technical staff. We are happy to help you determine your optimal

storage needs.

7. Reagents Required But Not Included

PCR Amplification

AmpliTaq Gold DNA Polymerase or equivalent (RECOMMENDED) (Life Technologies, Cat# N808-0241)

EagleTaq DNA Polymerase or equivalent (RECOMMENDED) (Roche Cat# 05206944190)

ABI Fluorescence Detection

HI-DI Formamide with ROX size standards - ABI 310 (IVS, Cat# 6-098-0051)


8. Recommended Positive Controls

Master Mix Target Color Control DNA Cat# Product Size in Base

Pairs

TCRG - 6FAM

Vg1-Vg11

+ Jg1/Jg2, JgP,

JgP1/JgP2

Blue

Specified Size Range 5% TCRG Positive Control DNA

---

4-088-3320 159-207

194, 196

Specimen Control

Size Ladder

Multiple Genes

Blue

TCRG Negative Control DNA

4-092-0020

84*, 96, 200, 300, 400,

600 Note: The amplicon sizes listed above were determined using an ABI 3130 platform. Amplicon sizes seen on your specific CE instrument may differ by

1-4 base pairs from those listed above depending on the platform of detection (ABI) and the version of the analysis software used. Once identified, the amplicon size as determined on your specific platform should be consistent from run to run. This reproducibility is extremely useful when tracking previously

identified clonal populations. *An 84 base pair peak may be detected and is most likely the result of primer-primer interaction.

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9. Procedure Notes

Autoclaving does not eliminate DNA contamination.

Work flow in the PCR laboratory should always be in a one way direction between separate work areas;

beginning in Master Mix Preparation, moving to the Specimen Preparation, then to the Amplification and

finally to Detection.

1. Do not bring amplified DNA into the areas designated for master mix or specimen preparation.

2. Due to the analytical sensitivity of this test, extreme care should be taken to avoid the contamination of

reagents or amplification mixtures with samples, controls or amplified materials. All reagents should be

closely monitored for signs of contamination (e.g., negative controls giving positive signals). Discard

reagents suspected of contamination.

3. All pipettes, pipet tips and any equipment used in a particular area should be dedicated to and kept to that

area of the laboratory.

4. PCR trays, bases and retainers must to be decontaminated in 10% bleach and rinsed with distilled water

two separate times before returning them to the starting areas.

5. Sterile, disposable plastic ware should be used whenever possible to avoid RNase or cross-contamination.

10. Reagent Preparation

All unknown samples should be tested using the Specimen Control Size Ladder master mix. This is to

ensure that no inhibitors of amplification are present and the DNA is of sufficient quality to generate a

valid result.

All samples should be tested in duplicate.

Positive, negative and no template controls should be tested.

1. Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently

vortex to mix.

2. In containment hood or dead air box remove an appropriate aliquot to a clean, sterile microfuge tube (one

tube for each of the master mixes). Aliquot volumes should be 45 μL for each sample + 135 μL (3 x 45 µl)

for the positive, negative and no template controls. We recommend adding an additional 20 μL to correct

for pipetting errors.

3. Add the appropriate amount of either AmpliTaq Gold, EagleTaq or AmpliTaq DNA polymerase (0.25 μL

of either AmpliTaq Gold, EagleTaq or AmpliTaq @ 5 U/l per 50 μL total reaction volume) to each of the

master mixes 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.

11. Sample Preparation

Using any method of DNA extraction, extract the genomic DNA from unknown samples. Resuspend DNA to

final concentration of 10 g – 200 g per mL in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or molecular

biology grade water. 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. Testing sample

DNAs with the Specimen Control Size Ladder master mix will ensure that DNA of sufficient quality was

present to yield a valid result.

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12. Amplification

1. Aliquot 45 μL of the master mix/enzyme solutions into individual PCR wells or tubes.

2. 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.

3. Amplify the reactions using the following PCR program.

Standard Program

Step 1: 95C for 7 minutes

Step 2: 95C for 45 seconds



Step 5: Go to step 2; 34 more times

Step 6: 72C for 10 minutes

Step 7: 15C forever

Remove the amplification plate from the thermal cycler.

13. Detection

Available Template Amplification Controls

The Specimen Control Size Ladder master mix primers are labeled with a fluorescent dye (6-FAM). This

label is detected as BLUE using the differential fluorescence software. The amplicons produced with this

master mix are at ~100, 200, 300, 400 and 600 base pairs. Please note that as the result of primer-primer

interactions there may be an additional peak at ~84 base pairs. The products of this master mix should be

run separately from those generated by any other master mix.

ABI Fluorescence Detection with ABI 310, 3100 & 3130 instruments

1. In a new microcentrifuge tube, mix an appropriate amount (10 μL per reaction) of Hi-Di Formamide with

ROX Size Standardsa. Vortex well.

2. In a new 96-well PCR plate, add 10 μL of Hi-Di Formamide with ROX size standards to individual wells

for each reaction.

3. 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.

4. Cap or cover the PCR plate.

5. Heat denature the samples at 95 ºC for 2 minutes, then snap chill on ice for 5 minutes.

6. Prepare a sample sheet and injection list for the samples.

7. Run the samples on an ABI 310/3100/3130 capillary electrophoresis instrument according to its user

manualb.

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

results.

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ABI Fluorescence Detection with ABI 3500 instruments

Note: Due to instrument to instrument variation in the performance of the ABI 3500 platform, the amount of formamide,

sample and size standard listed in the protocol is intended to be a starting point. The protocol may need to be optimized for

specific ABI 3500 Platforms.

1. In a new microcentrifuge tube, mix an appropriate amount (9.5 μL per reaction) of Hi-Di Formamide with

LIZ Size Standardsa. Vortex well.

2. In a new 96-well PCR plate, add 9.5 μL of Hi-Di Formamide with LIZ size standards to individual wells

for each PCR reaction.

3. Transfer 0.5 μL of each PCR reaction to the wells containing Hi-Di Formamide with LIZ size standards.

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


5. Heat denature the samples at 95ºC for 3 minutes, then snap chill on ice for 5 minutes.


7. Run the samples on an ABI 3500 capillary electrophoresis instrument according to its user manualb.

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

results.

Note a: Please see Applied Biosystems’ accompanying product insert for mixing Hi-Di Formamide with size standards for different ABI instruments. Note b: As the samples are run on the machine, they are fractionated, detected and analyzed by the instrument. Runs are 20-24 minutes in duration. The

ABI capillary electrophoresis instruments routinely handle 2 runs per hour (for the 16- and 24-capillary instruments this is equal to 768 and 1152

samples per day, respectively) and automatically analyze and store data for review or comparison with other test results.

14. Interpretation and Reporting

Note: This assay is for research use only. Although positive results are highly suggestive of malignancy, these

assays are designed for Research Use Only and, if used in a clinical setting, should only be used in support of

diagnosis. Positive and negative results should be interpreted in the context of all clinical information and

laboratory test results. PCR based testing does not identify 100% of clonal cell populations; therefore, repeat

testing by Southern blot may be advisable to rule out clonality.

Expected Size of Amplified Products

Master

Mix

Target Color Control DNA Cat# Product Size

(bp)

TCRG-

6FAM

All V and J genes

Vg9 + Jg1/Jg2

Vg10 + Jg1/Jg2

Blue

Blue

Blue

Specified Size Range TCRG Negative Control DNA



---

4-092-0020

4-088-3320

4-088-3320

159-207

159-207

194

196

Specimen

Control

Size

Ladder

Multiple Genes

Blue

Any Human DNA

---

84*, 96, 200,

300, 400, 600

Note: The amplicon sizes listed above were determined using an ABI 3130 platform. Amplicon sizes seen on your specific CE instrument may differ 1-4

base pairs from those listed above depending on the platform of detection (ABI) and the version of the analysis software used. Once identified, the amplicon size as determined on your specific platform should be consistent from run to run. This reproducibility is extremely useful when tracking

previously identified clonal populations. *An 84 base pair peak may be detected and is likely the result of primer-primer interaction.

Results can be reported as “Positive” or “Negative” for “Detection of T cell receptor gamma chain gene rearrangement consistent with the presence of a clonal cell population”

1. Samples that fail to amplify following repeat testing should be reported as “A result cannot be reported

on this specimen because there was DNA of insufficient quantity or quality for analysis”.

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2. All assay controls must be examined prior to interpretation of sample results. If the controls do not yield

the correct results, the assay is not valid and the samples should not be interpreted.

The following describes the analysis of each of the controls and the decisions necessary based upon the results:

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

Positive: Possible contamination of all PCR amplification reactions. Do not continue with

the interpretation of results. Prepare fresh master mix and repeat amplification.

Negative: Continue with the analysis.

2. Positive Control: (This can also be an extraction control if positive control material is taken through

extraction processes). If the positive control is:

Positive: Continue with analysis.

Negative: Repeat assay unless specimen tests positive.

3. Specimen Control Size Ladder: (This is run on unknown samples only). If the amplification control is:

Positive: ~100, 200, 300, 400 and 600 base pair products are seen. Because smaller PCR

fragments are preferentially amplified, it is not unusual for the 600 base pair

fragment to have a diminished signal or to be missing entirely. Continue with

analysis.

Negative: Repeat assay unless specimen tests positive.

Sample Interpretation Following the acceptance of the controls, the clinical samples are interpreted as follows:

Note: Criteria for defining a positive peak are as follows:

Products generated from samples that fall within the 159-207 base pair size range and are at least three times

the amplitude of either adjacent peak that might be present in the polyclonal background are consistent with a

positive peak.

One or two prominent peaks within the specified size range (159-207 base pairs) are reported as:

“Detection of T cell receptor gamma gene rearrangement(s) consistent with the presence of a clonal cell

population.”

Note: The size of amplified products can vary from instrument to instrument by 1-3 base pairs. 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 should be sequenced to confirm

their identity.

15. Limitations of Procedure

The assay is subject to interference by degradation of DNA or inhibition of PCR due to heparin or other agents.

The assay cannot reliably detect less than 5 positive cell per 100 normal cells.

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16. References

Greiner, TC, Rubocki, RJ. Effectiveness of Capillary Electrophoresis Using Fluorescent-Labeled Primers in

Detecting T-Cell 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 Laboratory-Developed 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, van Dongen, 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.

17. Appendix

Reagents and Special Supplies

Differential Fluorescence Detection



HI-Deionized Formamide (IVS, Cat# 6-098-0041)

HI-Deionized Formamide (Applied Biosystems, Cat# 4311320)

GS ROX 50-400HD Size Standard (Applied Biosystems, Cat# 402985)

18. Trouble Shooting Guide

Our laboratories are located in San Diego, California. Technical assistance is most rapidly obtained using our

Internet site: http://www.invivoscribe.com or by sending an email inquiry to: [email protected].

Alternatively, you can call (858) 224-6600 for technical assistance and information on our testing kits between the

hours of 8:00 AM and 5:00 PM Pacific Standard Time.

19. Symbols

The following symbols are now used in labeling for Invivoscribe NGS diagnostic products.

Catalog Number Expiration Date

Reagent Volume Authorized Representative in

the European Community

Lot Number Consult Instructions for Use

Storage Conditions For In Vitro Diagnostic Use

Manufacturer

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20. Sample Data

ABI Fluorescence Detection

The data shown below was generated using the TCRG-6FAM Master Mix. Amplified products were run on an ABI

3130 instrument.

Panel 1 displays data generated testing the 100% TCRG Positive Control DNA.

Panel 2 displays data generated testing a 5% dilution of the TCRG Positive Control DNA.

Panel 3 displays data generated testing the TCRG Negative Control DNA.

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21. TCRG Gene Rearrangement Assay 2.0: Single Page Flow Chart

1. Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to

mix.

2. In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube

for each of the master mixes). Aliquot volumes should be 45 μL for each sample + 135 μL for the positive,

negative and no template controls. We recommend adding an additional 20 μL to correct for pipetting errors.

3. Add the appropriate amount of either AmpliTaq Gold, EagleTaq or AmpliTaq DNA polymerase (0.25 μL of

either AmpliTaq Gold, EagleTaq or AmpliTaq @5 U/μL per 50 μL total reaction volume) to each of the master

mixes and gently mix by inverting several times or gently vortexing.

4. Aliquot 45 μL of master mix to individual wells of a PCR plate.

5. Add 5 μL of DNA from the unknown and control samples to individual tubes or wells containing the respective

master mix reactions and pipette up and down several times to mix. Amplify target DNA using the universal

thermal cycler program.

ABI Fluorescence Detection with ABI 310, 3100 & 3130 instruments

1. In a new microcentrifuge tube, mix an appropriate amount (10 μL per reaction) of Hi-Di Formamide with ROX

Size Standards. Vortex well.

2. In a new 96-well PCR plate, add 10 μL of Hi-Di Formamide with ROX size standards to individual wells for

each reaction.

3. 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.


5. Heat denature the samples at 95 ºC for 2 minutes, then snap chill on ice for 5 minutes.


7. Run the samples on an ABI 310/3100/3130 capillary electrophoresis instrument according to its user manual.

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

ABI Fluorescence Detection with ABI 3500 instruments

Note: Due to instrument to instrument variation in the performance of the ABI 3500 platform, the amount of

formamide, sample and size standard listed in the protocol is intended to be a starting point. The protocol may need

to be optimized for specific ABI 3500 Platforms.

1. In a new microcentrifuge tube, mix an appropriate amount (9.5 μL per reaction) of Hi-Di Formamide with LIZ

Size Standards. Vortex well.

2. 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.

3. 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.


5. Heat denature the samples at 95ºC for 3 minutes, then snap chill on ice for 5 minutes.


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

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

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