Instructions for Use

BCL2/JH t(14;18) 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: -85ºC to -65ºC(DNA controls may be separated from assay kits and stored at 2°C to 8°C)

Catalog#

13090010

Products

BCL2/JH t(14;18) Translocation Assay for Gel Detection
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Quantity

30 Reactions

Page 2 of 17

Table of Contents
1.

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

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

3.

PRINCIPLES OF THE PROCEDURE ............................................................................................................................................................. 3
3.1.
3.2.

4.

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.

8.

Thermal cycler .................................................................................................................................................................................. 6
Electrophoresis Unit ........................................................................................................................................................................ 6
UV Illumination Unit (for Gel Detection) ........................................................................................................................................ 6

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

7.

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

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

6.

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

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

5.

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

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

INTERPRETATION AND REPORTING ........................................................................................................................................................ 12
8.1.
8.2.

Sample Analysis ..............................................................................................................................................................................12
Sample Interpretation ...................................................................................................................................................................12

9.

LIMITATIONS OF PROCEDURE............................................................................................................................................................... 13

10.

EXPECTED RESULTS ........................................................................................................................................................................... 13
10.1.
10.2.

Expected Size of Amplified Products ............................................................................................................................................13
Sample Data ...................................................................................................................................................................................14

11.

TECHNICAL AND CUSTOMER SERVICE..................................................................................................................................................... 15

12.

REFERENCES .................................................................................................................................................................................... 1513.

SYMBOLS ........................................................................................................................................................................................ 15

14.

LEGAL NOTICE ................................................................................................................................................................................. 16
14.1.
14.2.

15.

Warranty and Liability....................................................................................................................................................................16
Patents and Trademarks ................................................................................................................................................................16

BCL2/JH T(14;18) TRANSLOCATION ASSAY: SINGLE PAGE GUIDE ............................................................................................................... 17

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 3 of 17

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

2. Summary and Explanation of the Test
2.1.

Summary
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 toan increase in the levels of B cells in the body.

2.2.

Background
This BCL2 t(14;18) Translocation Assay contains five (5) master mixes. Primers in four of the master mixes target thejoining region of the immunoglobulin heavy chain gene and distinct regions of the BCL2 gene. These master mixes
are used to detect BCL2 t(14;18) translocations. The remaining master mix targets a ubiquitous HLA class II gene toensure DNA of sufficient quality and quantity was present to generate a valid result. Positive and negative control
DNAs are also included.

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 the conserved 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 notcompletely 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.

Simplified view of the genomic organization of the BCL2 and IGH genes on chromosomes 18 and 14, respectively. Yellow
boxes represent the exon regions of the BCL2 gene. Exons of the immunoglobulin heavy chain gene are represented inother colors. The solid black lines represents intron regions, which have been left incompletely spliced to assist in
demarcation of the exon segments. Mbr and mcr type t(14;18) translocations are shown in the lower portions of the
figure with the relative positions of primers and the size of the amplicons generated from the positive control DNA
indicated.

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 4 of 17

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.

4. Reagents
4.1.

Reagent Components

Table 1. Available Kits

Catalog #13090010

Description

Total Reactions

BCL2/JH t(14;18) Translocation Assay for Gel Detection

30 Reactions

Table 2. Reagent Components for Gel Detection

Reagent

Reagent Components(active ingredients)

Catalog #Unit of
Quantity# of Units

1500 µL

11500 µL

11500 µL

11500 µL

11500 µL

1

100 µL1

100 µL1

100 µL

1

Storage Temp.

BCL2/JH t(14;18) (Mbr) Mix 1a – Unlabeled23090030

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 t(14;18) (Mbr) Mix 1b – Unlabeled23090010

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

Master Mixes
BCL2/JH t(14;18) (mcr) Mix 2a – Unlabeled23090040

Oligonucleotides targeting the 3’ minor cluster region(mcr) of the BCL2 gene and the J region of the IGH genein a buffered salt solution.

BCL2/JH t(14;18) (mcr) Mix 2b – Unlabeled23090020

Template
Amplification
Control Master
Mix

Amplification Control – Unlabeled20960010

40881750
Controls andStandard

Oligonucleotides targeting the minor cluster region(mcr) of the BCL2 gene and the J region of the IGH genein a buffered salt solution.

40881810
40880490

Oligonucleotides targeting the HLA-DQα locus; theMajor Histocompatibility Complex or Human Leukocyte
Antigen (HLA), class II, DQ alpha 1.

IVS-0030 Clonal Control DNA

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

IVS-0031 Clonal Control DNA

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

IVS-0009 Clonal Control DNA

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

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic proceduresor

280283 Rev. I | September 2023

Page 5 of 17

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 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 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
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.
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.
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 determine
optimal storage needs.

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 6 of 17

5. Instruments
5.1.

Thermal cyclerUse or Function: Amplification of DNA samples
Suggested Instrument: Veriti™ Thermal Cycler or equivalent
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 35 V to 135 V 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Capable of emitting light at a wavelength of ~302 nm

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

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 7 of 17

6. Specimen Collection and Preparation
6.1.

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

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 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
This is a robust assay system. A wide range of DNA concentrations will generate a valid result. Quantifying andadjusting DNA concentrations is generally not necessary.
6.4.1.
6.4.2.


6.5.

Using any method of DNA extraction, extract the gDNA from unknown samples.
Resuspend DNA to final concentration of 100 µg – 400 µg per mL in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or
distilled water.
When testing unknown samples, a template amplification control master mix (e.g., Amplification Control orSpecimen Control Size ladder) can be used to verify the absence of PCR inhibitors and sufficient quality andquantity of DNA is present to generate a valid result.

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

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 8 of 17

7. Assay Procedure
7.1.

Materials Provided
Please see Table 2 for a list of provided materials.

7.2.

Materials Required (not provided)

Table 3. Materials Required (not provided)

Reagent/Material

DNA Polymerase

Recommended Reagents/Materials and Suppliers
Roche:• EagleTaq DNA Polymerase
Invivoscribe, Inc.
• FalconTaq DNA Polymeraseor equivalentPart Number
05206944190
60970130

Notes

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
0.5 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, or PTC-240
Perkin-Elmer• PE 2600, 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/ Pyrogenfree

Microcentrifuge tubes

N/A

N/A

SterileThermo Fisher Scientific:• UltraPureTM 10 mg/mL Ethidium Bromide

15585-011

Agarose Gels

Thermo Fisher Scientific:• MetaPhorTM Agarose, 125 gor• LonzaTM NuSieveTM 3:1 Agarose

BMA50180
or
BMA50090

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

BCL2/JH t(14;18) Translocation Assay

N/A

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

N/A

N/A

N/A

N/A
Dilute 1:5 prior to use.
N/A
N/A
For agarose or polyacrylamidegels

280283 Rev. I | September 2023

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7.3.

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 remove an appropriate aliquot to clean, sterile microfuge tube (one tube
for each master mix).


7.3.3.

Add the appropriate amount of Taq DNA polymerase (0.25 μL @5 U/µL per 55 μL total reaction volume) toeach master mix and gently mix by inverting several times or gently vortexing.


7.3.4.

7.4.

Aliquot volumes are 50 μL for each sample + 150 μL (3 x 50 µL) for the positive, negative, and no templatecontrols.
Add an additional 20 μL to correct for pipetting errors.

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

Continue to step 7.4.1 for the single amplification protocol or step 7.4.5 for the nested amplification protocol.

Amplification

Single Amplification ProtocolStrategy:Advantage:

7.4.1.

Aliquot 50 µL of the master mix/enzyme solutions into individual PCR wells or tubes.


7.4.2.
7.4.3.

Employ two Master Mixes to independently identify BCL2 t(14;18) translocations involving the
major breakpoint and minor cluster regions (Mbr and mcr, respectively).
Two separate Master Mixes are used to identify Mbr and mcr BCL2 translocations.

BCL2/JH t(14;18) Mbr Mix 1b andBCL2/JH t(14;18) mcr Mix 2b

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 for EagleTaq

7.4.4.

Step

Temperature

Duration

Cycles1

95ᐤC

7 minutes

1

2

94ᐤC

30 seconds

3

55ᐤC

30 seconds

4

72ᐤC

1 minute

572ᐤC

10 minutes

1

6

4ᐤC

∞

1

35

Remove the amplification plate from the thermal cycler.

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 10 of 17

Nested Amplification ProtocolStrategy:Advantage:

7.4.5.

Aliquot 50 µL of the 1st round master mix/enzyme solutions into individual PCR wells or tubes.


7.4.6.
7.4.7.
7.4.8.
7.4.9.

BCL2/JH t(14;18) Mbr Mix 1b andBCL2/JH t(14;18) mcr Mix 2b

For sample reactions, add the diluted amplification products to the following master mixes:



7.5.

Mix thoroughly prior to adding to the prepared 2nd round amplification mixes

Repeat steps 7.4.1 - 7.4.4 for the 2nd round of amplification master mixes.



Note:

BCL2/JH t(14;18) Mbr Mix 1a andBCL2/JH t(14;18) mcr Mix 2a

Add 5 L of sampleor 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 appropriate PCR program in Table 4.
Remove the amplification plate from the thermal cycler.
Dilute the products from the first round of amplification 1:100 (2 μL + 198 μL of molecular biology grade
distilled water).


7.4.10.

Employ an outer set of primers to amplify Mbr and mcr of BCL2 t(14;18) translocation targets.
Followed by secondary amplification with an internal primer set to detect final product.
Better sensitivity if translocation is identified.

BCL2/JH t(14;18) Mbr Mix 1a product added into BCL2/JH t(14;18) Mbr Mix 1b
BCL2/JH t(14;18) mcr Mix 2a product added into BCL2/JH t(14;18) mcr Mix 2b

Detection

Available Template Amplification Controls
The Amplification Control master mix primers are available unlabeled for gel detection and labeled with afluorescent dye (6-FAM) for ABI fluorescence detection. The 6-FAM label is detected as BLUE using the differential
fluorescence software. The amplicons produced with this master mix are at 235 base pair (bp).
Run the products of this master mix separately.

Gel Detection – Agarose TBE Gels (RECOMMENDED)
7.5.1.
7.5.2.
7.5.3.
7.5.4.
7.5.5.

Prepare a 2% MetaPhor or NuSieve 3:1 agarose/TBE gel.
Individually mix 20 µL from each amplification reaction with 4 µL of 6X gel loading buffer.
Load 20 µL of this mixture into separate wells of the gel, flanked by DNA size standards.
Detect products using ethidium bromide or an equivalent dye.
Photograph the gel and interpret the data.

Gel Detection – Polyacrylamide TBE Gels
7.5.6.
7.5.7.
7.5.8.
7.5.9.

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.
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.

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 11 of 17

7.5.10.
7.5.11.
7.5.12.
7.5.13.

7.6.

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 with UV illumination.
Photograph the gel and interpret the data.

Recommended Positive Controls

Table 5. Recommended positive controls and size of amplified products

Master Mix

TargetControl DNA

Catalog #Product Size (bp)BCL2/JH t(14;18) Mbr
Mix 1aOutside BCL2
Mbr primers

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

550-800~800

BCL2/JH t(14;18) Mbr
Mix 1b

Inside BCL2 Mbrprimers

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

80-300215

BCL2/JH t(14;18) mcr
Mix 2aOutside BCL2
mcr primers

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

600-1200~1050

BCL2/JH t(14;18) mcr
Mix 2b

Inside BCL2 mcr
primers

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

500-1100~850

HLA-DQα

Valid Size Range
IVS-0009 Clonal Control DNA--40880490

235
235

Amplification Control

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

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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
More than 85% of all clonal B cell populations are identified with this assay. However, due to the diversity of theantigen receptor genes and heterogeneity in translocation breakpoints, subsequent testing of negative samples by
NGS may be advisable to rule out clonality.
Results can be reported as “Positive” or “Negative” for “Detection of BCL2 t(14;18) translocation”
8.1.1.
8.1.2.
8.1.3.

Samples that fail to amplify following repeat testing can be reported as “A result cannot be reported on thisspecimen 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 of each control and the decisions necessary based upon the results.


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.

o

Note:

There are several background peaks observed when testing the Polyclonal Negative Control and many normal
samples, see detailed description, above.
oPositive Control:ooPositive:
Negative:

Amplification Control:
o

o
8.2.

Negative:

235 bp products:
No peaks:

Continue with the analysis.
(This can also be an extraction control if positive control material is taken
through extraction processes). If the positive control is:Continue with analysis.
Repeat assay.
(This is run on unknown samples only). If the amplification control generates:
Continue with analysis.
Repeat assay and/or obtain a new specimen.

Sample Interpretation

Following the acceptance of the controls, the samples are interpreted as follows:
8.2.1.

BCL2/JH t(14;18) (Mbr) Mix 1a:
Presence of a prominent band within the 550 - 800 bp range is reported as: “Detection of a BCL2 t(14;18)
Translocation involving the major breakpoint region of BCL2.”

8.2.2.

BCL2/JH t(14;18) (Mbr) Mix 1b:
Presence of a prominent band within the 80 - 300 bp range is reported as: “Detection of a BCL2 t(14;18)
Translocation involving the major breakpoint region of BCL2.”

8.2.3.

BCL2/JH t(14;18) (mcr) Mix 2a:
Presence of a prominent band within the 600 - 1200 bp range is reported as: “Detection of a BCL2 t(14;18)
Translocation involving the minor cluster region of BCL2.”

8.2.4.

BCL2/JH t(14;18) (mcr) Mix 2b:
Presence of a prominent band within the 500 - 1100 bp range is reported as: “Detection of a BCL2 t(14;18)
Translocation involving the minor cluster region of BCL2.”

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 13 of 17

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

10. Expected Results
10.1.

Expected Size of Amplified Products
The size range for each master mix has been determined testing Polyclonal Negative Control DNAs. For accurate andmeaningful interpretation, it is important to ignore products that occur outside of the valid size range. Products that
occur outside of the expected size range are not indicative of BCL2 translocations.
Note:

Products may display slightly different mobility in different gels and buffers. Therefore it is important tovalidate with the system that you intend to use for routine analysis.

Table 6. Expected Size of Amplified Products

Master Mix

TargetBCL2/JH t(14;18) Mbr Mix
1a

Outside BCL2

BCL2/JH t(14;18) Mbr Mix
1b

Inside BCL2 Mbr

BCL2/JH t(14;18) mcr Mix 2b3Note:

Note:

4

Product Size (bp)--40880490
40881750

550-800
None1~800

Valid Size Range
IVS-0009 Clonal Control DNA
IVS-0030 Clonal Control DNA--40880490
40881750

80-300
None2
215

Valid Size Range
IVS-0009 Clonal Control DNA
IVS-0031 Clonal Control DNA--40880490
40881810

600-1200
None3
~1050

primers

Valid Size Range
IVS-0009 Clonal Control DNA
IVS-0031 Clonal Control DNA--40880490
40881810

500-1100
None4
~850

HLA-DQα

Any Human DNA

primers
Outside BCL2
mcr primers
Inside BCL2 mcr

Amplification Control
Note:
1Note:
2
Note:

Cat#

Valid Size Range
IVS-0009 Clonal Control DNA
IVS-0030 Clonal Control DNA

Mbr primers

BCL2/JH t(14;18) mcr Mix 2a

Control DNA

---

235

The amplicon sizes listed above were determined using an ABI platform.
Products of 150 bp and 500 bp may be observed testing the negative control and normal samples.
Products of 116 bp and 190 bp may be observed testing the negative control and normal samples. In nested applications amplicons at 525 bp
and 625 bp may be observed.
Product of ~600 bp may be observed testing the negative control and normal samples.
Product of ~325 bp may be observed testing the negative control and normal samples. In nested applications amplicons at 525 bp and 625 bp
may be observed.

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 14 of 17

10.2.

Sample Data

Gel Detection: The data shown in Figure 2 - Figure 6 were generated using the master mixes indicated.

Figure 2.

Amplified products were generated with the BCL2/JH
t(14;18) Master Mix 1a then run on a 2% agarose gel.

Figure 3.

Amplified products were generated with the BCL2/JH
t(14;18) Master Mix 1b then run on a 2% agarose gel.

Figure 4.

Amplified products were generated with the BCL2/JH
t(14;18) Master Mix 2a then run on a 2% agarose gel.

Figure 5.

Amplified products were generated with the BCL2/JH
t(14;18) Master Mix 2b then run on a 2% agarose gel.

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 15 of 17
These images were generated with IVS-0000 Polyclonal Control DNA; however, IVS-0009 clonal control DNA is also negative for BCL2/JH t(14;18)translocations and will yield similar results when used as the negative control.

Figure 6.

This is a photograph of a 2% agarose/TAE gel withdata generated from two different genomic DNA
samples (lanes 2 and 3). Both samples generate abright band at 235 bp.

11. Technical and Customer Service
Thank you for purchasing our BCL2/JH t(14;18) Translocation Assay. 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 inyour 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. Deininger, MWN, Goldman, JM and JV Melo. Blood, 2000, 96:3343-3356.
2. Miller, JE (Application filed: US Patent and Trademark Office)3. Miller, JE, Wilson, SS, Jaye, DL and Kronenberg, M. Molecular Diagnosis, 1999, 4: 101-117.

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

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 16 of 17

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 iscovered by product specifications and does not perform as specified, our policy is to replace the product or credit the full purchaseprice. No other warranties of any 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 arisingfrom the use, results of use, or inability to use its products; product efficacy under purchaser-controlled conditions in purchaser’slaboratory must be established and continually monitored through purchaser defined and controlled processes including but notlimited to testing of positive, negative, and blank controls every time a sample is tested. Ordering, acceptance, and use of productconstitutes 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 andfuture applications. All of these patents and applications are licensed exclusively to Invivoscribe®. Additional patents licensed to
Invivoscribe covering some of these products 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 purchase of 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 t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023

Page 17 of 17

15. BCL2/JH t(14;18) Translocation Assay: Single Page Guide
Single Amplification Protocol:
15.1.

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

15.2.

BCL2/JH t(14;18) Mbr Mix 1b andBCL2/JH t(14;18) mcr Mix 2b

In a containment hood or dead air box, remove an appropriate aliquot to clean, sterile microfuge tube (one tube foreach master mix).
Aliquot volumes are 50 μL for each sample + 150 μL for the positive, negative and no template controls.
Add 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 55 μL total reaction volume) to each mastermix and gently mix by inverting several times or gently vortexing.

15.4.

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

15.5.

Amplify the reactions using the PCR program in Table 4.

15.6.

Remove the amplification plate from the thermal cycler.

Nested Amplification Protocol:
15.7.

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

15.8.

Follow steps 15.2 - 15.6 using the following master mixes:



15.9.

BCL2/JH t(14;18) Mbr Mix 1a andBCL2/JH t(14;18) mcr Mix 2a

Dilute each product from the 1st round reactions 1:100 (2 μL + 198 μL of molecular biology grade distilled water) andmix prior to addition to the 2nd round Master Mixes.

15.10. Using gloved hands, remove the 2nd round master mixes from the freezer. Allow the tubes to thaw; then gently vortex
to mix.

15.11. Follow steps 15.2 - 15.6 using the following master mixes:


Note:

BCL2/JH t(14;18) Mbr Mix 1b andBCL2/JH t(14;18) mcr Mix 2b
For sample reactions, add the diluted amplification products to the following master mixes:

BCL2/JH t(14;18) Mbr Mix 1a product added into BCL2/JH t(14;18) Mbr Mix 1b
BCL2/JH t(14;18) mcr Mix 2a product added into BCL2/JH t(14;18) mcr Mix 2b

Gel Detection – Agarose TBE Gels
15.12. Prepare a 2% MetaPhor or NuSieve 3:1 agarose/TBE gel.
15.13. Individually mix 20 μL from each amplification reaction with 4 μL of 6X gel loading buffer.
15.14. Load 20 μL of this mixture into separate wells of the gel, and flank with DNA size standards.
15.15. Detect products using ethidium bromide or an equivalent dye.
15.16. Photograph the gel, interpret the data.

BCL2/JH t(14;18) Translocation Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280283 Rev. I | September 2023