LM DNA Kit Product Insert

LM DNA Kit Product Insert
Immucor Transplant Diagnostics, Inc.
550 West Avenue, Stamford, CT 06902 USA
Tel: (203) 328-9500 or (888) 329-0255, Fax: (203) 328-9599
Product Documentation and Translations available at: www.Immucor.com
For In Vitro Diagnostic Use
Definition of Symbols……………………..
Reagents by Catalog Number…………...
Intended Use…………………………………
Directions for Use…………………………
Summary and Explanation……………….
Principles of the Procedure………….....
C. Hybridization…………………………..
D. Analyze sample with Luminex
A. Identification…………………………..
B. W arnings and Cautions………………
C. Storage Instructions………………….
D. Purification or Treatment for Use....
E. Instability Indications…………………
Instrument Requirements………………..
Specimen Collection and Preparation..
Quality Control…………………………….
Limitation of the Procedure…………….
Expected Values…………………………..
A. Materials Provided……………………
Specific Performance characteristics.
Limited License……………………………
Trademarks Used………………………….
Appendix A………………………………….
Gel Electrophoresis……………………..
Gel Interpretation………………………..
B. Materials required, but not Provided... 4
C. Additional Materials to be Provided
by the User………………….................
A. Purify Genomic DNA…………………
B. Amplification………………...............
DEFINITION OF SYMBOLS (Product Labels and Supplemental Documents)
Batch Code
Upper Limit of
Use By
Keep away
from light
Sufficient for N
Do Not Freeze
Caution –
for Use
for use
in the European
Page 1 of 9
LC1437CE.3 (05/15)
LCT -N LIFECODES HLA-Null Allele SSO Typing Kit
Product #628939
LC-N LIFECODES HLA-Null Allele Typing Kit for use with Luminex
Product # 629100-50
Product Number
Fill volume
LIFECODES Null Allele Class 1
Master Mix
870 µL
2 to 8°C
LIFECODES Null Allele Class 2
Master Mix
870 µL
2 to 8°C
LIFECODES Null Allele Probe Mix
810 µL x 2
2 to 8ºC
Protect From Light
Dilution Solution
19.7 mL
18 to 30ºC
LIFECODES Taq Polymerase
25 µL x 2
-10ºC to -30ºC
Sufficient for 50 tests
Probe Mixes are light sensitive: keep exposure to light at a minimum.
CAUTION: Do not use components past their expiration dates.
CAUTION: Deviations from recommended protocol and required materials including LIFECODES Taq Polymerase have not been
DNA Typing of Class I and Class II Alleles in conjunction with LIFECODES HLA SSO Typing kits.
DNA-based HLA typing using PCR amplified DNA is a common laboratory procedure. PCR amplification of DNA is used as the
means to enrich for a selected DNA region. For HLA typing, a subsequent assay is utilized to determine the properties of the
amplified DNA. Several types of assays, such as SSP (1), direct SSOP (2), RFLP (3) and reverse SSOP dot blot technologies
(4), have been used in HLA typing. Like SSOP and reverse dot blot methods, LIFECODES HLA-SSO Typing kits utilize
sequence-specific oligonucleotides (SSOs) to identify which HLA alleles are present in a PCR amplified sample. It is the set of
SSOs employed, not the methodologies that determines the ability to distinguish among the various alleles present in the PCR
amplification. Whereas reverse dot blot and SSOP methods employ enzyme labels and colorimetric substrates that require
subsequent development, the LIFECODES assay is a homogenous multiplex system. That is, all SSOs are analyzed
simultaneously and the entire assay is carried out in a single reaction vessel with the addition of a single reagent.
The LIFECODES HLA-SSO Typing procedure is based on the hybridization of labeled single stranded PCR product to SSO
probes. Amplification of DNA using PCR typically employs equimolar amounts of both forward and reverse primer to generate a
double-stranded DNA product. However, if the amount of one primer is in excess relative to the other, the reaction will generate
some single-stranded DNA product in addition to double-stranded product. During the initial cycles of the LIFECODES
amplification step, double-stranded DNA is generated. Once the limiting primer is exhausted, the remaining primer uses the
double-stranded product as a template for generation of single-stranded DNA. This method generates both double stranded and
single stranded products that upon denaturation, will both participate in the hybridization reaction.
Each of the different probes may be homologous to a sequence within the amplified DNA that is unique to an allele or group of
alleles. In other words, these probes are designed so that each probe preferentially hybridizes to a complementary region that
may or may not be present in the amplified DNA. In addition, the amplified DNA is also hybridized to one or more Consensus
probes homologous to sequences present in all the alleles of a locus. SSO Typing can be affected by the type of biological
material, method of purification, amount and integrity of genomic DNA. Therefore, the signal obtained for the Consensus
probe(s) can serve as an indicator of the success of the amplification and hybridization procedures. Also, the signal obtained
with the Consensus probe can be used to normalize the signal of the allele specific probes and correct for variations in the
amount of amplified product in the hybridization reaction. The analysis of the results generated from the SSO typing can be used
to determine the presence or absence of particular DNA sequences in amplified DNA and to identify the possible alleles in the
For the LIFECODES HLA-SSO Typing procedure, probes are attached to Luminex Microspheres designed for use with the
Luminex Instrument. Up to 100 different populations of Luminex Microspheres can be mixed together and analyzed by the
Luminex Instrument because each population of microspheres can be distinguished by its unique fluorescence signature or
color. A different SSO probe can be attached to each color microsphere. Therefore, a mixture of several probes can be
distinguished from each other by virtue of their association with particular color microspheres. The Luminex Instrument is also
able to quantify the relative amounts of labeled PCR product hybridizing to each Luminex Microsphere. Therefore, the relative
signal obtained with the SSO probes in the LIFECODES assay, as with other SSOP methods, can be used to assign the probes
Page 2 of 9
LC1437CE.3 (05/15)
as having positive or negative reactivity with the amplified DNA sample (see Results section). This in turn provides the
information needed to determine the HLA phenotype of the sample.
Null 1 is used as a complementary assay to increase the resolution of LIFECODES HLA-A, HLA-B, HLA-C assays, solving the
allelic ambiguity between A*24:02/24:09N, B*51:01/ 51:11N, and C*04:01/04:09N. Null 2 is used as a complementary assay to
increase the resolution of LIFECODES HLA-DRB3,4,5 assay, solving the allelic ambiguity between DRB4* 01:03/ DRB4*
01:03:01:02N, and DRB5*01:02/DRB5*01:08N.
Merging of the information from the Null 1 or 2 assay with the corresponding locus-specific LIFECODES SSO Typing kit assay
results to generate a suggested typing of a sample can be conducted through manual analysis or software analysis. For manual
analysis, the output of the Null 1 or 2 assays identifies the presence of absence of specific null alleles. The result from the
corresponding locus-specific LIFECODES SSO typing kits may carry ambiguities involving these null alleles (e.g.,
A*24:02/24:09N, B*51:01/ 51:11N, C*04:01/04:09N, DRB4* 01:03/ DRB4* 01:03:01:02N, or DRB5*01:02/DRB5*01:08N). If the
null allele is identified with the Null product, all allele combinations that do not include the null allele can be eliminated from the
results obtained from the corresponding locus-specific LIFECODES SSO typing kit. If the null allele is absent based on the Null
product result, all allele combinations that include the Null allele can be eliminated from the results obtained from the
corresponding locus-specific LIFECODES SSO typing kit. In the product insert and in the software, this process of combining the
results of the two products is referred to as merging the results.
A. Identification
See tables in Reagents by Catalog Number section for complete listing of catalog numbers.
B. Warnings or Cautions
1. For In Vitro Diagnostic Use.
2. Separate pipettes should be designated for Pre-PCR manipulations as well as for Post-PCR manipulations.
3. Biohazard: All biological and blood samples should be treated as potentially infectious. Use Universal
Precautions when handling.
4. Dilution Solution, Probe mixes, TAQ Polymerase and R-Phycoerythrin Conjugated Streptavidin contain
hazardous compounds. Avoid contact with skin and eyes and dispose of all materials after use according to
local regulations. See Material Safety Data Sheets for additional information.
C. Storage Instructions
1. Refer to kit component packaging label for proper storage temperatures.
2. Probe mixes and R-Phycoerythrin Conjugated Streptavidin are light sensitive, KEEP FROM LIGHT; DO NOT
3. Do not use components past their expiration date.
D. Purification or Treatment Required for Use
See “Specimen Collection and Preparation.”
E. Instability Indications
1. If salts have precipitated out of solution during shipping or storage, resolubilize completely prior to use by
vortexing at room temperature (18 to 30°C).
2. Do not use R-Phycoerythrin Conjugated Streptavidin that has been frozen during shipment or storage.
1. Luminex Instrument and XY Platform (Product Number 888300, 888310)
2. The following Thermal Cyclers have been validated: 96-Well GeneAmp® PCR System 9700 set to MAX mode (Base Cat #
N8050200, Gold Block Cat #4314878), Veriti™ 96-Well Thermal Cycler set to 9700 MAX mode (Cat #4375786). Refer to
Table 2 for maximum ramp speeds. Caution: Other thermal cyclers and ramp speeds have not been validated.
A. Human DNA can be purified from Whole blood, Buffy coats and Buccal swabs using a validated method that meets the
criteria below. DNA extracted from blood preserved in EDTA and ACD (Acid Citrate Dextrose) have been tested and
shown to perform in this assay.
B. DNA extracted from blood preserved in heparin cannot be used in this assay. Other preservatives have not been
C. The isolated DNA should be in 10 mM TRIS, pH 8.0-9.0, or in nuclease free water. If a chelating agent such as EDTA is
present the final concentration of the chelating agent should not exceed 0.5 mM.
D. The presence of alcohol, detergents or salts may adversely affect DNA amplification.
E. Final DNA concentration should be 10 to 200 ng/µL.
F. Absorbance measurements of the DNA sample at 260 and 280nm should give a ratio of 1.65 to 2.0.
G. DNA can be used immediately after isolation or stored at –20ºC for up to 1 year. Repeated freeze/thawing should be
avoided since this can result in DNA degradation.
Page 3 of 9
LC1437CE.3 (05/15)
Caution: Deviations from recommended protocol and required materials have not been validated
A. Materials Provided (See tables in Reagents by Catalog Number section for specific information)
Appropriate Master Mix (MX)
Appropriate Probe Mix (BM)
Dilution Solution (DS)
Threshold Table(s), Probe Hit Chart(s)
No 628075) x 2
B. Materials, Required, but Not Provided
The following materials were used in the validation of the kit:
Luminex Sheath Fluid (1x LIFECODES Cat. No.
Nuclease-free water (LIFECODES Cat. No. 757003;
PCR tubes and caps - Corning Thermowell Tube
Strips (Costar® Cat. No. 6542, LIFECODES Cat. No.
888640) or Corning® Thermowell PCR 96 well plates
(Cat. No. CLS6551) or Themoscientific AB Gene®
Superplate 96-well PCR plate (Cat. No. AB-2100)
Costar® plate (Costar® Cat. No. 6509, LIFECODES
Cat. No. 888630)
Thermowell Clear Polyethylene Tape (Costar® No.
6524 (LIFECODES Cat. No. 888635)
R-Phycoerythrin Conjugated Streptavidin (SA-PE),
1mg/mL (LIFECODES Cat No. 628511)
Luminex Calibration Kits (Luminex 100/200 Calibration
Kit, Luminex 100/200 Performance Verification Kit,
LIFECODES Cat. Nos. 628018 and 628019
C. Additional materials to be provided by the user
Vortex Mixer
Silicone compression Mat. Axygen Scientific # CM-FLAT or equivalent
Bath Sonicator
Barrier filter tips
Pipettors, Multichannel pipettors and tips (1-20µL, 20-200µL,1000µL)
Spreadsheet analysis software
Heat Block
70% Isopropanol or 20% Bleach
Retainer tray – Applied Biosystems #403081 (for use with the 9700 thermal cycler only)
• Probe mixes and SA-PE are light sensitive: keep away from light and do not freeze.
• Warm beads at 55º to 60ºC for at least 5-10 minutes to thoroughly solubilize components in probe mixture.
• Sonicate briefly (~15 sec), then vortex probe mix for about 15 seconds to thoroughly suspend the beads.
• Take extreme caution in the aliquoting process, using calibrated pipettes. Failure to do so may result in reagent loss
and sample failure.
• All temperatures must be precisely maintained. Fluctuations as little as +/- 0.5°C can affect results.
• At the hybridization stage, samples should not remain in the diluted state at 56°C for more than 5 minutes (see Results
section). It is recommended to assay the amplified samples as soon as possible. If the samples cannot be run on the
Luminex Instrument the same day, the amplified product can be stored up to 3 days at 2-8ºC prior to use. For
longer storage, store at –20ºC up to one week until ready to assay. The amplified product can only be frozen
and thawed once. Repeated freezing and thawing will result in degradation of amplified samples and will yield
poor results if assayed.
A. Purify genomic DNA, using method of choice; final concentration should be 10 to 200 ng/µL. Adjust, if necessary,
with nuclease free water. Keep all samples at similar concentrations.
B. DNA amplification (PCR)
1. Allow the Master Mix to warm to room temperature (18 to 30°C).
2. Gently vortex for approximately 10 seconds. This will ensure the salts are in solution. Spin briefly (5 – 10 seconds) in
microcentrfuge to bring contents to the bottom of the tube.
3. Using Table 1 below, prepare the components for amplification for n+1 reactions using the indicated amount of each
component per reaction (except for DNA). Bring to a final volume of 20µL per reaction with nuclease free water. Gently
4. Pipette the appropriate amount of Genomic DNA (40 to 120ng) into the PCR tubes.
5. Aliquot the amplification mix into the PCR tubes containing the genomic DNA. (The total volume of amplification mix and
genomic DNA should equal 20µL for each sample reaction.)
6. Cap tubes tightly to prevent evaporation during PCR.
Page 4 of 9
LC1437CE.3 (05/15)
7. Place samples in the thermal cycler and run program, see Table 2 and 3.
Table 1. Reaction Components for Amplification
Amount per PCR sample reaction
Genomic DNA 10-200ng/µL
Total of ~80ng
LIFECODES Taq Polymerase
0.2µL (1U)
Nuclease-free water
To 20µL final volume
Table 2. Thermal Cycler Conditions for Amplification
Thermal Cycler
GeneAmp® PCR System 9700
Veriti™ 96-Well Thermal Cycler
Mode (Ramp speed)
MAX mode
9700 MAX mode
Table 3. Thermal Cycler Conditions for Amplification
Temperature and Incubation Time
# of Cycles
95º C for 3 min
95º C for 15 sec
60º C for 30 sec
72º C for 30 sec
95º C for 10 sec
63º C for 30 sec
72º C for 30 sec
72º C for 2 min
4º C forever
Note: To be sure of sample amplification, refer to Product Gel Electrophoresis (Appendix A).
C. Hybridization
Be sure hybridization buffer components of the LIFECODES probe mix are solubilized and that the beads are
thoroughly suspended.
Turn on the Luminex Instrument and XY Platform to allow for 30 minute warm-up.
1. Warm probe mix in a 55º to 60ºC heat block for at least 5 -10 minutes to thoroughly solubilize components in probe
2. Sonicate briefly (~15 sec), then vortex probe mix for about 15 seconds to thoroughly suspend the beads.
3. Combine 15 µL of the appropriate probe mix with 5 µL of locus specific PCR product into each well of a thermal cycler 96 well plate
(Costar® No. 6509). When aliquoting probe mix to more than 10 wells, gently vortex probe mix after each set of ten. Seal plate with
polyethylene tape (Costar® No. 6524).
Place silicone compression mat on top of plate prior to hybridization.
5. Hybridize samples under the following incubation conditions:
Table 4. Thermal Cycler Conditions for Hybridization
97ºC for 2 minutes
47ºC for 10 minutes
56ºC for 8 minutes
Ensure that the detection laser on the Luminex Instrument is turned on at least 30 minutes before the
hybridization ends.
6. While the samples are hybridizing, prepare a 1:200 SA-PE mixture / Dilution Solution. Combine 170µL Dilution Solution
(DS) and 0.85 µL 1mg/mL SA-PE per sample. It is recommended to make enough Dilution Solution Mixture for n+1
samples to account for pipetting loss. (See Table 5)
7. Keep Dilution Solution/ SA-PE mixture in the dark, at room temperature; SA-PE is light sensitive! The Dilution Solution
may be warmed at 45ºC for 5 minutes and vortexed upon arrival to ensure all components are in solution. Dilution
Page 5 of 9
LC1437CE.3 (05/15)
solution must be at room temperature (18 to 30°C) before making the mixture. Prepare prior to use and discard any
remaining portion.
Table 5. Dilution Solution Preparation Volumes
# of Samples
Dilution Solution (DS)
8. At the 56ºC hold, while the tray is on the thermal cycler, dilute each sample with 170µL of the prepared Dilution Solution/
SA-PE mixture. It is critical to dilute all samples within 5 minutes (following the 8 minute 56°C HOLD step).
9. Remove the sample tray from the thermal cycler and place in the Luminex Instrument.
D. Analyze sample using the Luminex Instrument*
For best results, assay the samples immediately using the Luminex Instrument.
1. Turn on the Luminex Instrument between 30 minutes and 4 hours before assaying the samples.
2. Prior to analyzing the samples on the Luminex Instrument, set up a Batch Run by which the samples will be analyzed.
a) Select Create a New Batch from the File menu.
For example, if analyzing for Null Class I, add Batch for Null Class I
The Batch Template is provided on website and is named, in this case, Null1 xxxxxx (lot#).
Please note that the template versions are lot number specific and correspond to the probe mix lot numbers.
Follow the stepwise instructions that appear on the screen for creating batches.
When naming the batch, do not include commas in the name because information after a comma will be
lost upon exportation of the data.
For further instructions on creating batches and multibatches, refer to the Luminex User’s Manual
b) Click the eject icon to eject the plate holder. Place the 96 well thermal cycler plate containing the samples in the
XYP heater block present on the plate holder.
c) Click the Retract icon. The samples are now ready to be analyzed. A prime step should be performed before
starting the run.
d) After the samples have been run through the instrument, a sanitization step with 70% Isopropanol or 20% household
bleach should be performed followed by two wash steps. The instrument can be turned off at this point if it is not
going to be used for the remainder of the day.
3. After a batch is complete, the data is exported as a comma separated values (csv) file. These files are named
‘OUTPUT.CSV’ and saved in a folder with the Batch Name. This data is then available for making typing assignments as
described below.
*Refer to Luminex User’s Manual for instrument operation, including daily startup, calibration, maintenance, and shutdown
Sample typing can be done as follows:
The generated CSV files can be opened and the data processed with common spreadsheet programs such as Microsoft Excel,
Lotus 123, Corel Quattro Pro, or similar software. Analysis is comprised of the following steps:
1) Verify that the Number of Events for each SSO in each sample is at least 60. This information is found in the DataType:
Count section of the CSV file.
2) Determine that the values for the Consensus probes for each sample are above their minimum Median Fluorescent
Intensity or MFI. The minimum thresholds are lot specific and can be found in the Threshold Table.
• To obtain reliable results, there must be sufficient data gathered by the Luminex Instrument.
• Collect at least 60 events for each SSO.
3) Subtract the Background Control value for each probe from the sample values producing the background corrected data
set. Background Control values are found in the Threshold Table and are lot specific. Background values are average
MFI values for each bead to compensate for background noise due to bead variation.
Page 6 of 9
LC1437CE.3 (05/15)
4) For each sample, divide the background-corrected data for each probe by the background-corrected value for the
corresponding consensus probe producing the normalized data set.
MFI (Probe) – MFI (Control blank for probe)
MFI (Consensus) – MFI (Control blank for consensus)
5) For each probe, record the normalized value on the Threshold Table Worksheet.
6) Once all values have been assigned, the probe hit pattern (i.e., the combination of all positive and negative assignments
for a given sample) can be compared with the Probe Hit Chart (LC1024) provided on website.
• There is a separate threshold table for each locus.
• These threshold tables are Lot-specific; be certain that the Lot # on the threshold tables matches the Lot # in the
typing kit.
• If a normalized value for a particular probe falls above the maximum threshold for a negative assignment and below the
minimum value for a positive assignment, the sample should be considered as indeterminate for this probe. The sample
should be typed, first assuming the value to be negative and then again assuming the value to be positive.
• See EXPECTED VALUES section for further information on threshold values.
It is recommended that one negative and positive control be run with each test, such as a water blank and a previously typed
sample respectively. Consensus SSO probes, listed on the Threshold Table, hybridize to their respective locus specific alleles.
Values obtained with the Consensus SSOs from positive controls should exceed the threshold value for the SSO as set forth in
the Threshold Table Worksheet.
The LIFECODES Probe Mix(es) contain one or more consensus SSO probes identified in the typing kit worksheets. These
consensus probes hybridize to all alleles and act as internal controls to verify amplification and to confirm that hybridizations
occurred. If the minimum value is not obtained for these SSOs, the sample may not produce the correct typing and the sample
test should be repeated.
The assay should be run as recommended in the package insert as well as performed with any other quality control procedures
that are in accordance with local, state, federal and/or accreditation agencies requirements.
The PCR conditions and assay conditions described require precisely controlled conditions. Deviations from these parameters
may lead to product failure.
All instruments must be calibrated according to manufacturer’s recommendations and operated within manufacturer’s prescribed
1) Beads must be pre-warmed and well suspended prior to use. This ensures that the hybridization buffer components are in
2) 47ºC and 56ºC incubations require a high degree of accuracy (+/- 0.5ºC). A thermal cycler should be employed.
Temperature should be verified, within wells of the 96 well thermal cycler plate, using a thermocouple (e.g., Bio-Rad, Model
VPT-0300 or equivalent). The temperature within wells and among wells should not vary more than +/- 0.5ºC.
3) Time at 56ºC is critical and should not exceed a total of 13 minutes. This includes the 8-minute incubation plus no more than
5 minutes to dilute all the samples with Dilution Solution/ SA-PE mixture.
4) Once diluted, sample analysis must be complete within 1 hour (protect from light).
5) Do not mix components from other kits and lots.
Due to the complex nature of HLA typing, qualified personnel should review data interpretation and typing assignments.
Page 7 of 9
LC1437CE.3 (05/15)
Low Bead Count
Probe Mix not well suspended
Instrument not
functioning properly
Prewarm, sonicate and vortex probe mix and repeat
Calibrate Instrument. (Refer to Luminex IS User’s
Remove and sonicate needle. Perform backflush.
Call Immucor Transplant Diagnostics, Inc. if problem
persists. (888) 329-0255
Check DNA concentration and purity.
Salts in Master Mix are out of
Heat Master Mix at 37ºC for 5 minutes, vortex gently
and spin down briefly.
Poor Taq Polymerase
Use validated LIFECODES Taq Polymerase Catalog #
Run Thermal profile on Thermal cycler to verify
parameters are within specified parameters.
Warm dilution solution at 45ºC for 5 minutes before
use and vortex. Store at room temperature.
Replace R-Phycoerythrin Conjugated Streptavidin.
Out of Calibration
Sample flow path blocked
CON Threshold
Sample failed to amplify
or amplified poorly*
Amplification conditions not within specific parameters
Low Median Fluorescent Intensity Value (MFI)
Multiple SSO
failures or sample
fails to yield a HLA
typing result
Allele specific
Amplification conditions not
within specific parameters
Run Thermal profile on Thermal cycler to verify
parameters are within specified parameters.
DNA sample contaminated
Re-isolate DNA from Blood sample.
DNA partially degraded
Evaporation during hybridization step
If not using an entire plate, leave one row empty on
each side of samples to be assayed to allow plate to
be sealed tightly.
* PCR amplification can be verified by gel electrophoresis (See Appendix A).
Each Locus has one CON probe and two SSO probes. If a sample contains one of the loci being assayed, the consensus probe
and at least one of the SSO probes for that locus should be positive. Probe values typically can be resolved as positive and
negative. In some rare instances, a value may fall between the positive and negative cutoff values and therefore is considered to
be indeterminate. If a sample contains indeterminate values for a particular SSO probe, the sample should be typed with the
probe as a negative and again with the probe as a positive. If two SSO probes for a locus are indeterminate, the sample cannot
be typed and should be re-assayed.
As noted in the Limitations of the Procedure section, it is critical to precisely follow the protocol. Any deviations can
lead to sample typing failure.
When LIFECODES HLA SSO Null Allele Typing Kits are used according to the procedure described in the product insert, the
Class I and Class II HLA type of DNA samples can be determined. The HLA -Null Allele Class 1 (Null1) assay shows 100%
agreement (97.4% lower boundary of 95% Confidence Interval) for A locus, 100% agreement (97.4% lower boundary of 95%
Confidence Interval) for B locus, and 100% agreement (97.4% lower boundary of 95% Confidence Interval) for C locus in 139
samples evaluated when compared to results obtained with bi-directional sequencing. The HLA -Null Allele Class 2 (Null2)
assay shows 97.1% agreement (92.7% lower boundary of 95% Confidence Interval) for DRB 4 locus, and 98.5% agreement
(94.8% lower boundary of 95% Confidence Interval) for DRB 5 locus in 137 samples evaluated when compared to results
obtained with bi-directional sequencing.
1. Olerup, O., et al. (1992) Tissue Antigens 39:225
2. Saiki, RK., et al. (1986) Nature 324: 163
3. Maeda, M., et al. (1989) Tissue Antigens 34: 290
4. Bugawan, TL., et al. (1990) Immunogenetics 32: 231
Taq polymerase is manufactured for Immucor Transplant Diagnostics by Promega Corp. It is licensed to Promega under U.S.
Patent Nos. 5,338,671 and 5,587,287 and their corresponding foreign patents. The purchase of this product includes a limited,
non-transferable license under U.S. patent 5,981,180 or its foreign counterparts, owned by Luminex Corporation, to perform
multiplex analysis of clinical specimens for HLA typing.
Page 8 of 9
LC1437CE.3 (05/15)
Immucor Transplant Diagnostics, Inc., 550 West Avenue, Stamford, CT 06902 USA.
Phone: 203-328-9500, 888-329-0255 Fax: 203-328-9599
Authorized Representative:
Immucor Medizinische Diagnostik GmbH , Adam-Opel-Strasse 26A Rodermark 63322, Germany
Phone: (+49) 6074-84 20 -0, Fax: (+49) 6074-84 20-99
European Technical Service: Phone: +32/3 385 4791
This document last revised and issued: Rev: 3, 2015-05-15
AB Gene®
IDNA™ Agarose
AB Gene House
Corning Incorporated
Bio-Rad Laboratories, Inc.
Lonza Group, Ltd.
Lonza Group, Ltd.
Gene Amp®
Luminex Corporation
Roche Molecular System
Applied Biosystem
Immucor Inc.
Gel Electrophoresis
The PCR reactions performed in the LIFECODES HLA-SSO Typing Kits are designed to produce both double and single
stranded products, which are the predominant products that hybridizes to the SSOs. For quality assurance or to trouble shoot an
experiment it might be necessary to perform gel electrophoresis to examine the PCR reaction for the presence of amplified DNA.
Materials Required (as listed or equivalent)
Electrophoresis Grade Agarose (Lonza Group, Ltd.
IDNA® Agarose No. 50170)
Electrophoresis apparatus/power supply
1X Gel Buffer (40xTAE, Promega No. V4281)
GelStar® Nucleic Acid Gel Stain (Lonza Group, Ltd. No.
UV Transilluminator (ChromatoVUE, UVP Inc. Model
Photographic imaging system
The relative migration of the single stranded product is dependent upon the gel concentration and buffer system employed.
Approximate migrations for each amplification are listed below for samples run in a 2% Agarose gel in 1X TAE buffer.
Electrophoresis Conditions
1. Remove GelStar® Nucleic Acid Stain (Lonza Group, Ltd. No 50535) from freezer to thaw. Keep in dark.
2. The gel used for this procedure must be 2%, i.e. for a 200ml gel bed use 4 grams of agarose to 200mL 1X TAE (Dilute from
40X TAE). Add 10µL GelStar® Nucleic Acid Stain to the molten agarose. When pouring the gel be sure to leave ample
room for DNA to run a significant distance (1 to 2 inches). USE CAUTION: GelStar® is a potential Carcinogen.
NOTE: It is possible to run gels with 20µL of 10mg/mL Ethidium Bromide in place of GelStar® Nucleic Acid Stain. Product band
intensity will be less in gels containing Ethidium Bromide than in gels containing GelStar®. USE CAUTION: Ethidium Bromide
is a known Carcinogen.
3. Keep gel in dark and allow to solidify.
4. Load a mixture of 2.5µL of each PCR product and 2.5µL 2X loading buffer with visible dye per sample, per amplification. Let
gel run in the dark at approximately 160 volts for 45 minutes or until sample runs far enough to see separate bands for
single and double stranded product (bromophenol blue band or other visible marker migrates 1 to 2 inches from wells).
5. Photograph using UV Transilluminator accompanied by a GelStar® Yellow Photographic Filter (Cambrex No 50536).
CAUTION: Wear protective equipment when handling GelStar® Nucleic Acid Stain or Ethidium Bromide and when
photographing gel using UV Transilluminator.
6. Gel analysis
Double Strand(s) (bp)
Single Strand(s) (bp)
Gel Interpretation
Double Stranded DNA
Single Stranded DNA
--------(less bright)
Primer Band
Page 9 of 9
LC1437CE.3 (05/15)
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF