Instructions for Use

BCL1/JH Translocation Assay
For Identification of BCL1/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 #13080010
13080020

Products
BCL1/JH Translocation Assay for Gel Detection
BCL1/JH Translocation Assay MegaKit for Gel Detection

Quantity
33 Reactions
330 Reactions

Page 2 of 14

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.

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.

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.

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

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

ASSAY PROCEDURE................................................................................................................................................................ 8
7.1.
Materials Provided .................................................................................................................................................................. 8
7.2.
Materials Required (not provided).......................................................................................................................................... 8
7.3.
Reagent Preparation ............................................................................................................................................................... 9
7.4.
Amplification ........................................................................................................................................................................... 9
7.5.
Detection............................................................................................................................................................................... 10
Gel Detection – Agarose TBE Gels ........................................................................................................................................................ 10
Gel Detection – Polyacrylamide TBE Gels ............................................................................................................................................ 10
7.6.
Quality Control ...................................................................................................................................................................... 10
7.7.
Recommended Positive Controls .......................................................................................................................................... 10

8.

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

Sample Reporting .................................................................................................................................................................. 11
Sample Interpretation ........................................................................................................................................................... 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 ....................................................................................................................................... 12

12.

REFERENCES....................................................................................................................................................................... 1313.

SYMBOLS .......................................................................................................................................................................... 13

14.

LEGAL NOTICE .................................................................................................................................................................... 13
14.1.
14.2.

15.

Warranty and Liability ........................................................................................................................................................... 13
Patents and Trademarks ....................................................................................................................................................... 13

BCLI/JH TRANSLOCATION ASSAY: SINGLE PAGE GUIDE................................................................................................................ 14

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 3 of 14

1. Assay Use
This Research Use Only assay identifies BCL1/JH t(11;14) translocations and is useful for the evaluation of new research andmethods in malignancy studies.

2. Summary and Explanation of the Test
2.1.

Background
This aberrant BCL1/JH t(11;14) translocation juxtaposes genes of the immunoglobulin heavy chain (IGH) gene on
chromosome 14q32 with the cyclin D1 gene on chromosome 11q13. The juxtaposition of IGH-sequences results inthe transcriptional activation of cyclin D1.2,3 Cyclin D1 is involved in the regulation of the G1 progression and G1/S
transition of the cell cycle.8 Translocation does not lead to expression of a fusion protein. In fact, oncogenesis is due
to a promoter/enhancer exchange, wherein the immunoglobulin gene enhancer stimulates the expression of cyclin
D1. Overexpression of cyclin D1, in turn, accelerates passage of transformed cells through the G1 phase.

2.2.

Summary
Invivoscribe’s gene rearrangement and translocation assays represent a simple approach to PCR-based clonality
testing. These standardized assays were carefully optimized testing positive and negative control samples using
multiplex master mixes. Included in this test kit are two (2) master mixes. The BCL1/JH master mix targets the majortranslocation cluster (MTC) of the BCL1 locus and the joining region of the Ig heavy chain locus. The other mastermix, the Specimen Control Size Ladder, targets multiple genes and generates a series of amplicons of 96, 197, 297,
397 and 602 base pair (bp) that can be used to ensure that the quality and quantity of input DNA is adequate to yielda valid result.
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 MTC regionof the BCL1/JH translocation and amplifies genomic DNA between primers that target the BCL1 gene and theconserved joining (JH) regions of the IGH gene (BCL1/JH Tube master mix). Breakpoints that occur outside the MTC
will not be identified by this particular test. Therefore, a negative result does not completely exclude the presenceof a BCL1/JH gene rearrangement in the sample.2 DNA from a normal lymphocyte population will also generate anegative result.

Figure 1.

Schematic diagram of the IGH-CCND1 t(11;14) translocation showing the cyclin D1 (CCND1) gene on the left and the Ig heavy
chain (IGH) gene on the right. Shown are relative positions and orientations for the BCL1/MTC primer and the JH primer, which
are included in the BCL1/JH Master Mix tube.

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 4 of 14

Gel Detection

3.2.

Gel electrophoresis, such as agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresis(PAGE), is commonly used to resolve the different 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 toeasily migrate through the gel matrix, whereas larger DNA fragments migrate more slowly. This causes a separationof the amplicon products based on size. Ethidium bromide or other DNA intercalating dyes can then be used to stain
and detect these products in the gel.

4. Reagents
Reagent Components

4.1.

Table 1. Available Kits

Catalog #Description

Total Reactions

13080010

BCL1/JH Translocation Assay for Gel Detection

33 Reactions

13080020

BCL1/JH Translocation Assay MegaKit for Gel Detection

330 Reactions

Table 2. Reagent Components

Reagent

Catalog #()

Reagent Components(active ingredients)

Unit of
Quantity

13080010# of Units

13080020# of Units

1500 µL

1

101500 µL

1

10100 µL

1

5

Storage Temp.

BCL1/JH Tube Master Mix
Master Mixes

23080010

Template
Amplification
Control MasterMix

20960020

40880550
Control andStandard

Oligonucleotides targeting the MTC of the BCL1
gene and the J region of the IGH gene in a bufferedsalt solution.

Specimen Control Size Ladder Master Mix

Multiple oligonucleotides targeting housekeeping
genes.

IVS-0010 Clonal Control DNA

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

or40920010

IVS-0000 Polyclonal Control DNA

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

100 µL

1

5

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

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 5 of 14

4.2.

Warnings and Precautions










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 and other 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.






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 comes 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 handle as directed. Do not use kitsbeyond their expiration date.
Adherence to the protocol will assure optimal performance and reproducibility. Ensure correct thermal cycler
programs are used, as other programs may provide inaccurate/faulty data such as false-positive and falsenegative results.
Dispose of unused reagents and waste in accordance with country, federal, state and local regulations.
Perform all laboratory procedures with standard personal protective equipment (gloves, laboratory coats andprotective eyewear). Follow good laboratory practices and universal precautions when working with specimens.
Do not pipette by mouth. Do not eat, drink or smoke in laboratory work areas. Wash hands thoroughly after
handling specimens and assay reagents. Handle specimens in approved biological safety containment facilities
and open only in certified biological safety cabinets. Use molecular biology grade water for the preparation of
specimen DNA.

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

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

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 6 of 14

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: 15ºC to 96ºC
Minimum Ramping Speed: 0.8ºC/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

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.

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 7 of 14

6. Specimen Collection and Preparation
6.1.

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

6.2.

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





6.3.

Divalent cation chelators
Low retention pipette tips
EDTA (not significant at low concentrations)
HeparinSpecimen Requirements 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.
Ship at ambient temperature; OR
Formalin-fixed paraffin embedded tissue or slides.

Sample Preparation


6.4.1.
6.4.2.

Test all samples in singlicate.
Include positive, negative, and no template controls with each master mix tested.
Using any method of DNA extraction, extract the gDNA from unknown samples.
Resuspend DNA to final concentration of 100 μg – 400 μg per mL in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or
distilled 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.


6.5.

When testing unknown samples, use a template amplification control master mix (e.g., Amplification
Control or Specimen Control Size ladder) 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.

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 8 of 14

7. Assay Procedure
7.1.

Materials Provided
See Table 2 for materials provided.

7.2.

Materials Required (not provided)

Table 3. Materials Required (not provided)

Reagent/Material

Ficoll Separation

Density GradientMediumRecommended Reagents/Materials and Suppliers
Thermo Fisher Scientific:• Ficoll Paque PREMIUM• 1X PBS diluted from 10X PBS• RPMI 1640
• Fetal Bovine Serum
Sigma:• DMSO Hybri-Max
Thermo Fisher Scientific®:

Part Number

Notes

45-001-752
70011-044
11875-093
16000-0XX**size dependent: 036 (100mL), 044 (500 mL), or 069(1000 mL)

D2650

Ficoll-PAQUE Premium

45-001-752

Buffered Saline Solution

Thermo Fisher Scientific:• 1X PBS diluted from 10X PBS

70011-044

Growth Medium

Thermo Fisher Scientific:• RPMI 1640 with L-glutamine

11875-093

Dimethyl sulfoxide

Sigma:• DMSO Hybri-Max

Fetal Bovine Serum

Thermo Fisher Scientific:• HyClone™ Fetal Bovine Serum (U.S.), Characterized

DNA Polymerase

Roche:• EagleTaq DNA Polymerase
Invivoscribe, Inc.
• FalconTaq DNA Polymeraseor equivalent

•

D2650
SH3007103
05206944190
60970130

N/A
N/A
N/A
N/A
N/A

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, PTC-200, PTC-220, 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/
Pyrogen-free

Microcentrifuge tubes

N/A

N/A

SterileEthidium Bromide

Agarose Gels

BCL1/JH Translocation Assay

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

15585-011

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

BMA50180
or
BMA50090

For RESEARCH USE ONLY; not for use in diagnostic procedures

N/A

N/A

280282 Rev. F | September 2023

Page 9 of 14
Table 3. Materials Required (not provided)

Reagent/Material

Recommended Reagents/Materials and SuppliersPart Number

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

Gel Electrophoresis Unit

N/A

N/A

Notes
N/A
Dilute 1:5 prior to use.
N/A
N/A
For agarose or polyacrylamide
gels

Reagent Preparation

7.3.



7.3.1.
7.3.2.

Test 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 positive, negative and no template controls with each master mix
Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex tomix.
In containment hood or dead air box, aliquot the appropriate volume of each master mix into a clean, sterile
microfuge tube.
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.


7.3.3.

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

Amplification

7.4.

7.4.1.
7.4.2.
7.4.3.

Aliquot 45 μL of the master mix/enzyme solutions into individual PCR wells or tubes.
Add 5 μL of sample or control DNA to the individual tubes or wells containing the respective master mix
reactions. Pipette up and down several times to mix.
Amplify the reactions using the PCR program in Table 4:
Use the calculated option for temperature measurement with the PTC instruments.



Table 4. Thermal cycling conditions

Program for EagleTaq (RECOMMENDED)
Step

Modified program

Temperature

Duration

CyclesStep

Temperature

Duration

Cycles1

95ᐤC

7 minutes

1

1

95ᐤC

7 minutes

1

2

95ᐤC

45 seconds

2

94ᐤC

30 seconds

3

60ᐤC

45 seconds

3

60ᐤC

45 seconds

4

72ᐤC

90 seconds

4

72ᐤC

120 seconds

5

72ᐤC

10 minutes

1

5

72ᐤC

10 minutes

1

6

15ᐤC

∞

1

6

15ᐤC

∞

1

7.4.4.

35

35

Remove the amplified PCR plate from the thermal cycler.

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 10 of 14

Detection

7.5.

Gel Detection – Agarose TBE Gels
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.

7.5.1.
7.5.2.



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.

7.5.3.
7.5.4.

Gel Detection – Polyacrylamide TBE Gels
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.

7.5.5.
7.5.6.



Load 20 μL of mixture into wells of the gel.

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

7.5.7.


7.5.8.
7.5.9.
7.5.10.
7.5.11.

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

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

Quality Control

7.6.

Run positive and negative, or normal controls (furnished with the kit) in singlicate each time the assay is performed toensure proper performance of the assay. Also include a no template control (e.g. water) to test for contamination. A
buffer control may also be added to ensure that no contamination of the buffer used to resuspend the samples has
occurred. The values for the positive controls are provided under section 10.1. Expected Size of Amplified Products.
Additional controls and sensitivity controls (dilutions of positive controls into our negative control) are available from
Invivoscribe.

Recommended Positive Controls

7.7.

The amplicon sizes listed were determined using an ABI platform or by gel electrophoresis.
Table 5. Recommended positive controls and size of amplified products

Master Mix

TargetBCL1/JH Tube

MTC of BCL1 + IGH JH

Specimen Control Size
LadderMultiple Genes

Note:
1Note:

Control DNA

Catalog #Product Size (bp)

Valid Size Range
IVS-0010 Clonal Control DNA--40880550

150 - 2000
~2001, 600

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

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

The amplicon sizes listed above were determined using a 2% agarose gel.
The ~200 bp band is comprised of a 174 bp and a 200 bp band. Both of these bands co-migrate at ~200 bp on an agarose gel.

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 11 of 14

8. Interpretation and Reporting
This assay is designed for Research Use Only, not intended for use in diagnostic procedures. PCR based testing does not
identify 100% of clonal cell populations; therefore, repeat testing by Next Gen Sequencing (NGS) may be advisable to ruleout clonality.

Sample Reporting

8.1.

Results can be reported as “Positive” or “Negative” for “presence of the BCL1/JH t(11;14) Translocation”.
8.1.1.
8.1.2.



Report samples that fail to amplify with the Specimen Control Size Ladder following repeat testing as “A result
cannot be reported on this specimen because there was DNA of insufficient quantity or quality for analysis”.
All assay controls must be examined prior to interpretation of sample results. The following describes theanalysis of each control and the decisions necessary based upon the results.
Negative Control:oPositive:o

Negative:

Positive Control:

o
o
o
o

8.2.

Possible contamination of all PCR amplifications. Do not continue with theinterpretation of results. Prepare fresh master mix and repeat amplification.
Continue with the analysis, provided that the polyclonal control is positive with theSpecimen Control Size Ladder.
(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 the assay.

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



Note:

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

Generates ~96, 197, 297, 397, and 602 bp products: Please note smaller PCR fragments are
preferentially amplified, and it is not unusual for the 602 bp fragment to have a diminishedsignal or to be missing entirely. Continue with analysis.
Generates no peaks:
Repeat the assay and/or obtain a new specimen.

Under suboptimal conditions a non-specific product of 550 bp is sometimes generated. To discriminate between
specific and non-specific products, the negative control DNA will not show this band. If a band is present in thenegative control, we then consider the band non-specific.

Sample Interpretation
Following the acceptance of the controls, interpret the samples as follows: One or two prominent bands within thevalid size range are reported as “Detection of a BCL1/JH t(11;14) 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.

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 12 of 14

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 andmeaningful interpretation, it is important to ignore peaks that occur outside of the valid size range for each mastermix. Peaks that are outside of the range cannot be assumed to be valid.

Table 6. Expected size of amplified products

Master Mix

TargetControl DNA

Catalog #Product Size (bp)--40920010
40880550---

150 - 2000
no product~2001, 600~5502--40920010

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

BCL1/JH Tube

MTC of BCL1 + IGH JH

Valid Size Range
IVS-0000 Polyclonal Control DNA
IVS-0010 Clonal Control DNA
Test samples

Specimen Control Size
LadderMultiple Genes

Valid Size Range
IVS-0000 Polyclonal Control DNA

Note:
1Note:
2Note:

10.2.

The amplicon sizes listed above were determined using a 2% agarose gel.
The ~200 bp band is comprised of a 174 bp and a 200 bp band. Both of these bands co-migrate at ~200 bp on an agarose gel.
The ~550 bp band is non-specific and may not be observed.

Sample Data

Figure 2.

The data shown above were generated with the
BCL1/JH Tube master mix.

Figure 3.

The data shown above were generated with theSpecimen Control Size Ladder master mix.

11. Technical and Customer Service
Thank you for purchasing our BCL1/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 yourlaboratory. 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

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 13 of 14

12. References
1.
2.
3.

van Dongen, JJM, et. al., (2003). 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. 17(12):2257-2317.
Janssen, J.W.G., et. al., (2000). Concurrent activation of a novel putative transforming gene, myeov and cyclin D1 in a
subset of multiple myeloma cell lines with t(11;14)(q13:q32). Blood. 95:2691-2698.
Shimazaki C, et. al., (1997). Over expression of PRAD1/cyclin D1 in plasma cell leukemia with t(11;14)(q13;q32). Int J
Hematol. 66(1):111-5.

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 product specifications
and does not perform as specified, our policy is to replace the product or credit the full purchase price. No other warranties of any kind, expressedor 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 to use its products; product efficacy underpurchaser-controlled conditions in purchaser’s laboratory must be established and continually monitored 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 useof 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. All of thesepatents 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 thesepatents 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 thetrademarks of others used herein) their respective owners.

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023

Page 14 of 14

15. BCLI/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 master mix).

Aliquot volumes are 45 μL for each sample + 135 μL for the positive, negative and no template controls.
Add an additional 20 μL to correct for pipetting errors.

15.3.

Add the appropriate amount of Taq DNA polymerase (0.25 μL @5 U/μL per 50 μL total reaction volume) to each mastermix 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 mastermix reactions and pipette up and down several times to mix.

15.6.

Amplify target DNA using the thermal cycler program below.
Program for EagleTaq (RECOMMENDED)

Step

Modified program

Temperature

Duration

CyclesStep

Temperature

Duration

Cycles1

95ᐤC

7 minutes

1

1

95ᐤC

7 minutes

1

2

95ᐤC

45 seconds

2

94ᐤC

30 seconds

3

60ᐤC

45 seconds

3

60ᐤC

45 seconds

4

72ᐤC

90 seconds

4

72ᐤC

120 seconds

5

72ᐤC

10 minutes

1

5

72ᐤC

10 minutes

1

∞

1

6

15ᐤC

∞

1

6

15ᐤC

35

35

Gel Detection – Agarose TBE Gels

15.7.

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

15.8.

Individually mix 20 μL from each amplified PCR product 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 the gel and interpret the 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. Voltage and run time can be adapted accordingly.
15.16. Stain the gels in 0.5 μg/mL Ethidium Bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.
15.17. Destain the gels 2X in water for 5-10 minutes.
15.18. Use UV illumination for visualization.
15.19. Photograph the gel and interpret the data.

BCL1/JH Translocation Assay

For RESEARCH USE ONLY; not for use in diagnostic procedures

280282 Rev. F | September 2023