Instructions for Use

BCL2/JH Translocation Assay

PCR Assay for identification of BCL2/JH translocations.
This assay is for Research Use Only. Not for use in diagnostic procedures.

Manufactured in U.S.A.

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

Catalog#

13090020
13090040

Products

BCL2/JH Translocation Assay for Gel Detection
BCL2/JH Translocation Assay MegaKit for Gel Detection

Quantity

33 reactions
330 reactions

Page 2 of 15

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 ...............................................................................................................................................................3
3.1.
3.2.

4.

Polymerase Chain Reaction (PCR) ............................................................................................................................................................ 3
Gel Detection ........................................................................................................................................................................................... 4

REAGENTS ..........................................................................................................................................................................................4
4.1.
4.2.
4.3.

5.

Reagent Components............................................................................................................................................................................... 4
Warnings and Precautions ....................................................................................................................................................................... 5
Storage and Handling ............................................................................................................................................................................... 5

INSTRUMENTS .....................................................................................................................................................................................6
5.1.
5.2.
5.3.

6.

Thermal cycler.......................................................................................................................................................................................... 6
Electrophoresis Unit ................................................................................................................................................................................. 6
UV Illumination Unit ................................................................................................................................................................................ 6

SPECIMEN COLLECTION AND PREPARATION ................................................................................................................................................7
6.1.
6.2.
6.3.
6.4.
6.5.

7.

Precautions .............................................................................................................................................................................................. 7
Interfering Substances ............................................................................................................................................................................. 7
Specimen Requirements and Handling .................................................................................................................................................... 7
Sample Preparation ................................................................................................................................................................................. 7
Sample Storage ........................................................................................................................................................................................ 7

ASSAY PROCEDURE ..............................................................................................................................................................................8
7.1.
7.2.
7.3.
7.4.
7.5.
7.6.
7.7.

8.

Materials Provided ................................................................................................................................................................................... 8
Materials Required (not provided) ........................................................................................................................................................... 8Reagent Preparation ................................................................................................................................................................................ 9
Amplification ............................................................................................................................................................................................ 9
Detection ............................................................................................................................................................................................... 10
Available Template Amplification Controls ............................................................................................................................................ 10
Recommended Positive Controls ........................................................................................................................................................... 11

INTERPRETATION AND REPORTING..........................................................................................................................................................11
8.1.
8.2.

Sample Interpretation ............................................................................................................................................................................ 11
Sample Reporting ................................................................................................................................................................................... 11

9.

LIMITATIONS OF PROCEDURE ................................................................................................................................................................11

10.

EXPECTED RESULTS.............................................................................................................................................................................12

10.1.
10.2.

Expected Size of Amplified Products...................................................................................................................................................... 12
Sample Data ........................................................................................................................................................................................... 12

11.

TECHNICAL AND CUSTOMER SERVICE ......................................................................................................................................................13

12.

REFERENCES .....................................................................................................................................................................................13

13.

SYMBOLS .........................................................................................................................................................................................14

14.

LEGAL NOTICE ...................................................................................................................................................................................14

14.1.
14.2.

15.

Warranty and Liability ............................................................................................................................................................................ 14
Patents and Trademarks ........................................................................................................................................................................ 14

BCL2/JH TRANSLOCATION ASSAY: SINGLE PAGE GUIDE ..............................................................................................................................15

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 3 of 15

1. Assay Use
This Research Use Only assay identifies BCL2/JH t(14;18) translocations.

2. Summary and Explanation of the Test
2.1.

Background

BCL2 translocations are reciprocal chromosome exchanges that place the bcl-2 proto-oncogene, located on chromosome 18,
under aberrant transcriptional control of the immunoglobulin heavy chain gene, located on chromosome 14. The bcl-2 protein
is an antagonist to apoptosis (programmed cell death), a normal process designed to eliminate unneeded and damaged cells
during hematopoiesis. Increased expression of the bcl-2 protein leads to an increase in the levels of B cells in the body.

2.2.

SummaryThis BCL2 t(14;18) Translocation Assay contains four (4) master mixes. Primers in three (3) of the master mixes target the joining
region of the immunoglobulin heavy chain gene and distinct regions of the BCL2 gene. These master mixes are used to detect
major breakpoint region (Mbr) and minor cluster region (mcr) of the BCL2 t(14;18) translocations. The remaining master mix,
the Specimen Control Size Ladder, targets multiple genes and generates a series of amplicons of 96, 197, 297, 397, and 602 basepairs (bp) to ensure DNA of sufficient quality and quantity was present to generate a valid result. Positive and negative control
DNA are also included.
This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.

3. Principles of the Procedure
3.1.

Polymerase Chain Reaction (PCR)

PCR assays are routinely used for the identification of chromosome translocations. This test targets the Mbr and mcr regions of
the BCL2/JH translocations (see Figure 1) and amplifies genomic DNA between primers that target the BCL2 gene and theconserved joining (J) regions of the IGH gene (BCL2/JH Tubes A, B, and C). Breakpoints that occur outside the Mbr and mcr
regions will not be identified by this particular test. Therefore, a negative result does not completely exclude the presence of a
BCL2/JH gene rearrangement in the sample.1 DNA from a normal lymphocyte population will also generate a negative result.

Figure 1.

Depicted is a schematic diagram of the BCL2/JH t(14;18) translocation showing the BCL2 gene on the left and the Ig heavy chain (IGH)
gene on the right. Shown are the relative positions and orientations for the major breakpoint region (Mbr) primers, the minor cluster
region (mcr) primers, and the JH primer, which are included in the 3 BCL2/JH master mix tubes.

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 4 of 15

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 is negatively charged,
when an electrical potential (voltage) is applied across the gel containing PCR products, the electrical 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 otherDNA intercalating dyes can then be used to stain and detect these products in the gel.

4. Reagents
4.1.

Reagent Components

Table 1. Available Kits

Catalog #Description

Total Reactions

13090020

BCL2/JH Translocation Assay for Gel Detection

33 Reactions

13090040

BCL2/JH Translocation Assay MegaKit for Gel Detection

330 Reactions

Table 2. Reagent Components

Reagent

Catalog #Reagent Components(active ingredients)

Unit of
Quantity

13090020# of Units

13090040# of Units

1500 µL

1

101500 µL

1

101500 µL

1

101500 µL

1

10

Storage Temp.

BCL2/JH Tube A – Unlabeled23090050

Multiple oligonucleotides targeting the major
breakpoint region (Mbr) of the BCL2 gene and the J
region of the IGH gene in a buffered salt solution.

BCL2/JH Tube B – Unlabeled
Master Mixes

23090060

Multiple oligonucleotides targeting the 3’ major
breakpoint region (3’ Mbr) of the BCL2 gene and the J
region of the IGH gene in a buffered salt solution.

BCL2/JH Tube C – Unlabeled23090070

Template
Amplification
Control Master
Mix

Positive
Controls

Negative(Normal)Control DNA

20960020

Multiple oligonucleotides targeting the minor cluster
region (mcr) of the BCL2 gene and the J region of the
IGH gene in a buffered salt solution.

Specimen Control Size Ladder – Unlabeled

Multiple oligonucleotides targeting housekeeping
genes.

40881750

IVS-0030 Clonal Control DNA
200 µg/mL of DNA in 1/10th TE solution

100 µL

1

540900070

IVS-P002 Clonal Control DNA
1600 pg/mL of plasmid DNA diluted in IVS-0000
polyclonal control DNA in 1/10th TE solution

100 µL

1

540881810

IVS-0031 Clonal Control DNA
200 µg/mL of DNA in 1/10th TE solution

100 µL1

5100 µL1

540920010

IVS-0000 Polyclonal Control DNA

200 µg/mL of DNA in 1/10th TE solution

or

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 5 of 15

4.2.

Warnings and Precautions



















4.3.

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 kits beyond
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 false-negative
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 safetycabinets. Use molecular biology grade water for the preparation of specimen DNA.
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 areasand 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 and othermaterials from post-PCR into the pre-PCR space.
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.

Storage and HandlingStore the assay at -85ºC to -65ºC until ready to use.
The optimum storage temperature for DNA controls is 2°C to 8°C, but DNA controls can be stored long term at 85°C to -65°C.
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.

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 6 of 15

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

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



5.3.

Minimum Thermal Range: 15°C to 96°C
Minimum Ramping Speed: 0.8°C/sec

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

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

UV Illumination Unit

Use or function: DNA detection
Performance characteristics and specification:oCapable of emitting light at a wavelength of ~302 nm

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

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 7 of 15

6. Specimen Collection and Preparation
6.1.

Precautions

Biological specimens from humans may contain potentially infectious materials. Handle all specimens in accordance with the
OSHA Standard on Bloodborne Pathogens 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 and HandlingThis assay tests 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
(stored at 2°C to 8°C and shipped at ambient temperature)
Formalin-fixed paraffin embedded tissue or slides (stored and shipped at ambient temperature)Sample Preparation

Extract the gDNA from specimens as soon as possible. Resuspend DNA to a final concentration of 100 µg to 400 µg per mL in
1/10th TE (1 mM Tris-HCl, pH 8.0; 0.1 mM EDTA) or in molecular biology grade or USP water. This is a robust assay system. A
wide range of DNA concentrations will generate a valid result. Therefore, quantifying and adjusting DNA concentrations isgenerally not necessary. Testing sample DNA with the Specimen Control Size Ladder master mix will ensure that DNA ofsufficient quality and quantity was present to yield a valid result.

6.5.

Sample Storage

Store gDNA at 2°C to 8°C or at -85°C to -65°C for long term storage.

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 8 of 15

7. Assay Procedure
7.1.

Materials Provided
Table 3: Kit components

7.2.

Catalog #Description

23090050

BCL2/JH Tube A – Unlabeled23090060

BCL2/JH Tube B – Unlabeled23090070

BCL2/JH Tube C – Unlabeled20960020

Specimen Control Size Ladder – Unlabeled

40881750

IVS-0030 Clonal Control DNA

40900070

IVS-P002 Clonal Control DNA

40881810

IVS-0031 Clonal Control DNA

40920010

IVS-0000 Polyclonal Control DNA

Materials Required (not provided)

Table 4: Materials Required (not provided)

Reagent/Material

DNA Polymerase

Glass Distilled De-ionized
Molecular Biology Grade or
USP Water
Calibrated Pipettes

Thermal cycler

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

Catalog #()
05206944190
60970130

Notes

N/A

N/A

Sterile and free of DNase andRNase.

Rainin:• P-2, P-20, P-200, and P-1000 pipettes• Or SL-2, SL-20, SL-200, and SL-1000 pipettes

N/A

Must be able to accurately
measure volumes between 1 µL
and 1000 µL.

Bio-Rad:• MJ Research PTC-100 or PTC-200, PTC-220, PTC-240
Perkin-Elmer• PE 9600 or PE 9700

N/A

N/A

N/A

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/Pyrogenfree

Microcentrifuge tubes

N/A

N/A

SterileGel Electrophoresis Unit

N/A

N/A

For polyacrylamide gels

Thermo Fisher Scientific:• UltraPure 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 BlueJuice Gel Loading Buffer• Novex Hi-Density TBE Sample Buffer (5X)

10816-015
LC6678Ethidium Bromide

BCL2/JH Translocation Assay

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

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 9 of 15
Table 4: Materials Required (not provided)

Reagent/Material

Recommended Reagents/Materials and Suppliers
Thermo Fisher Scientific:• TrackIt 100 bp DNA Ladder

100 bp DNA Ladder

7.3.

Notes
N/A

10488-058

Reagent Preparation

Test all unknown samples with the Specimen Control Size ladder master mix to ensure that no inhibitors of
amplification are present and there is DNA of sufficient quality and quantity to generate a valid result.
Test all samples in singlicate.
Test positive, negative and no template controls with each master mix.

7.3.1.

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

7.3.2.

In containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube
for each master mix).


7.3.3.

Aliquot volumes are 45 µL for each sample + 135 µL (3 x 45 µL) for the positive, negative and no templatecontrols.
Include 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 eachmaster mix and gently mix by inverting several times or gently vortexing.


7.4.

Catalog #()

The master mixes are now ready for distribution to reaction tubes or plate and addition of sample.

Amplification
7.4.1.

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

7.4.2.

Add 5 µL sample or control DNA to the individual tubes or wells containing the respective master mix reactions.


7.4.3.

Pipette up and down several times to mix.

Amplify the reactions using the following PCR program:Use the calculated option for temperature measurement with the PTC instruments.
Table 5: Thermal cycling conditions

7.4.4.

Step

Temperature

Duration

Cycles1

95°C

7 minutes

1

2

95°C

45 seconds

3

60°C

45 seconds

4

72°C

90 seconds

5

72°C

10 minutes

1

6

15°C

∞

1

35

Remove the amplification plate from the thermal cycler

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 10 of 15

7.5.

Detection

Gel Detection – Agarose TBE Gels (RECOMMENDED)
7.5.1.

Prepare a 2% MetaPhor or NuSieve 3:1 agarose/TBE gel.

7.5.2.

Individually mix 20 μL from each amplification reaction with 4 μL of 6X gel loading buffer.

7.5.3.

Load 20 μL of this mixture into separate wells of the gel, flanked by DNA size standards.

7.5.4.

Detect products using ethidium bromide or an equivalent dye.

7.5.5.

Photograph gel and interpret data.

Gel Detection – Polyacrylamide TBE Gels
7.5.6.

Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel and 0.5X TBE running buffer.

7.5.7.

Add 5 μL of ice-cold non-denaturing bromophenol blue loading buffer to samples.

7.5.8.

Load 20 μL of mixture into wells of the gel.

7.5.9.

Run gel at 110V for 2-3 hours or 40-50V overnight.
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.

7.5.10.

Gels are stained in 0.5 µg/mL ethidium bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.

7.5.11.

De-stain gels 2X in water for 5-10 minutes.

7.5.12.

Visualize gels using UV illumination.

7.5.13.

Photograph gel and interpret data.

Gel Detection – Heteroduplex Analysis
7.5.14.

Denature 20 μL PCR products at 94ºC for 5 minutes.

7.5.15.

Re-anneal PCR products at 4ºC for 60 minutes.

7.5.16.

Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel and 0.5X TBE running buffer.

7.5.17.

Add 5 μL ice-cold non-denaturing bromophenol blue loading buffer to samples.

7.5.18.

Load 20 μL of mixture into wells of the gel.

7.5.19.

Run gel at 110V for 2-3 hours or 40-50V overnight.



7.6.

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.

7.5.20.

Stain gels in 0.5 µg/mL ethidium bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.

7.5.21.

De-stain gels 2X in water for 5-10 minutes.

7.5.22.

Visualize gels using UV illumination.

7.5.23.

Photograph gel and interpret data.

Available Template Amplification Controls

The Specimen Control Size Ladder master mix generates amplicons at 96, 197, 297, 397 and 602 bp.


The ~100 bp band is comprised of 84 bp and 96 bp bands. Both of these bands co-migrate on a gel.
Run the products of this master mix separately.

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 11 of 15

7.7.

Recommended Positive Controls

Table 6: Recommended Positive ControlsaMaster Mix

TargetControl DNA

Catalog #Product Size in base pairs (bp)BCL2/JH Tube A

Mbr of
BCL2/JH

Valid Size Range
IVS-0030 Clonal Control DNA--40881750

100 - 2500
~250, ~750a

BCL2/JH Tube B

3’ Mbr of
BCL2/JH

Valid Size Range
IVS-P002 Clonal Control DNA--40900070

100 - 2500
139

BCL2/JH Tube C

mcr of
BCL2/JH

Valid Size Range
IVS-0031 Clonal Control DNA--40881810

100 - 2500
~390, ~800

Specimen Control
Size LadderMultiple
Genes

Valid Size Range
IVS-0000 Polyclonal Control DNA--40920010

84, 96, 197, 297, 397, 602
84, 96, 197, 297, 397, 602

Note:

The ~750 bp band (BCL2/JH Tube A) and the ~800 bp band (BCL2/JH Tube C) are generally weak bands.

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

8.1.

Sample Interpretation
8.1.1.

Samples that fail to amplify following repeat testing should be reported as “A result cannot be reported on thisspecimen because there was DNA of insufficient quantity or quality for analysis”.

8.1.2.

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 of each of the controls and the decisions necessary based upon the results.


Negative Control:oPositive:o

Negative:

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

Specimen Control Size Ladder:
o
o

8.2.

Positive:
Negative:(Polyclonal control, water or no template blank). If the negative control is:(This is run on unknown samples only). If the amplification control:

Generates 96, 197, 297, 397 and 602 bp products:
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. Continue with analysis.
Does not generate indicated products:
Repeat the assay and/or re-extract the specimen.

Sample Reporting

Following the acceptance of the controls, the samples are interpreted as follows: One or two prominent bands within the 100 2500 bp range are reported as “Detection of a BCL2/JH t(14;18) Translocation”.

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

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 12 of 15

10. Expected Results
10.1.

Expected Size of Amplified Products

The size range for each master mix has been determined testing positive control samples. For accurate and meaningfulinterpretation, ignore bands that occur outside of the valid size range for each master mix. Bands outside of the range cannot
be assumed to be valid.
Note:

These master mixes use a consensus JH primer. Therefore, a positive specimen can generate several valid products.
These amplicons differ in size by about 500 bp which is the distance between intron segments between thesetandem JH exons.

Table 7: Expected Size of amplified ProductsaMaster Mix

TargetControl DNA

Catalog #Product Size in base pairs (bp)BCL2/JH Tube A

Mbr of
BCL2/JH

Valid Size Range
IVS-0000 Polyclonal Control DNA
IVS-0030 Clonal Control DNA
IVS-0031 Clonal Control DNA--40920010
40881750
40881810

100 - 2500--~250, ~750a---

BCL2/JH Tube B

3’ Mbr of
BCL2/JH

Valid Size Range
IVS-0000 Polyclonal Control DNA
IVS-P002 Clonal Control DNA
IVS-0030 Clonal Control DNA
IVS-0031 Clonal Control DNA--40920010
40900070
40881750
40881810

100 - 2500
--138
-----

BCL2/JH Tube C

mcr of
BCL2/JH

Valid Size Range
IVS-0000 Polyclonal Control DNA
IVS-0030 Clonal Control DNA
IVS-0031 Clonal Control DNA--40920010
40881750
40881810

100 – 2500----~390, ~800

Specimen Control
Size LadderMultiple
Genes

Valid Size Range
Any Human DNA

Note:

10.2.

84, 96, 197, 297, 397, 602
84, 96, 197, 297, 397, 602---

The ~750 bp band (BCL2/JH Tube A) and the ~800 bp band (BCL2/JH Tube C) are generally weak bands.y.

Sample Data

The data shown below were generated using the master mix indicated. Amplified products were run on a 2% agarose gel.

Figure 2. BCL2/JH Tube A master mix.

Figure 3. BCL2/JH Tube B master mix.

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 13 of 15

Figure 4. BCL2/JH Tube C master mix.

Figure 5.

Specimen Control Size Ladder master mix.

11. Technical and Customer Service
Thank you for purchasing our BCL2/JH Translocation Assay. We appreciate your business. We are happy to assist you in thevalidation 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

12. References
1. van Dongen, JJM et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin
and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted
Action BMH4-CT98-3936. Leukemia. 2003, 17(12):2257-2317.

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 14 of 15

13. Symbols
The following symbols are used in Invivoscribe product labeling.
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.
©2023 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.

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 15 of 15

15. BCL2/JH Translocation Assay: Single Page Guide
15.1.

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

15.2.

In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube foreach of the master mixes).
Aliquot volumes are 45 μL for each sample + 135 μL for the positive, negative and no template controls.
Include an additional 20 μL to correct for pipetting errors.

15.3.

Add the appropriate amount of Taq DNA polymerase (0.25 μL @5 U/µL per 50 μL total reaction volume) to eachmaster mix and gently mix by inverting several times or gently vortexing.

15.4.

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

15.5.

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.

15.6.

Amplify target DNA using the universal thermal cycler program.

Gel Detection – Agarose TBE Gels (RECOMMENDED)
15.7.

Prepare a 2% MetaPhor or NuSieve 3:1 agarose/TBE gel.

15.8.

Individually mix 20 μL from each amplification reaction with 4 μL of 6X gel loading buffer.

15.9.

Load 20 μL of this mixture into separate wells of the gel, flanked by DNA size standards.

15.10.

Detect products using ethidium bromide or an equivalent dye.

15.11.

Photograph gel and interpret data.

Gel Detection – Polyacrylamide TBE Gels
15.12.

Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel and 0.5X TBE running buffer.

15.13.

Add 5 μL of ice-cold non-denaturing bromophenol blue loading buffer to samples.

15.14.

Load 20 μL of mixture into wells of the gel.

15.15.

Run gel at 110V for 2-3 hours or 40-50V overnight.

15.16.

Gels are stained in 0.5 µg/mL ethidium bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.

15.17.

De-stain gels 2X in water for 5-10 minutes.

15.18.

Visualize gels using UV illumination.

15.19.

Photograph gel and interpret data.

Gel Detection – Heteroduplex Analysis
15.20.

Denature 20 μL PCR products at 94ºC for 5 minutes.

15.21.

Re-anneal PCR products at 4ºC for 60 minutes.

15.22.

Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel and 0.5X TBE running buffer.

15.23.

Add 5 μL ice-cold non-denaturing bromophenol blue loading buffer to samples.

15.24.

Load 20 μL of mixture into wells of the gel.

15.25.

Run gel at 110V for 2-3 hours or 40-50V overnight.

15.26.

Stain gels in 0.5 µg/mL ethidium bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.

15.27.

De-stain gels 2X in water for 5-10 minutes.

15.28.

Visualize gels using UV illumination.

15.29.

Photograph gel and interpret data.

BCL2/JH Translocation Assay

280316 Rev. G | October 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures