SSP UniTray
SSP UniTray®
Instructions for Use
For Both Low and High Resolution Kits
SSP UniTray®
Instructions for Use
For In Vitro Diagnostic Use
The Invitrogen™ SSP UniTray® is a PCR-based method designed to provide low to high resolution
of the various HLA Class I and II types. Formulations of allele or group specific primer sets are
used to amplify genomic DNA using a 96 well thermal tray. Setup includes mixing a reaction
buffer with a human genomic DNA sample and Taq DNA Polymerase*, dispensing the mixture to
the UniTray®, sealing and then thermal cycling. After cycling is complete, the PCR products are
loaded onto a 2% agarose gel for electrophoresis. After electrophoresis, the ethidium bromide
stained gel is photographed and interpreted using a worksheet for the specific amplification
patterns. The test can be completed in 2.5 hours post DNA isolation. (Times vary depending on
make and model of the thermal cycler used).
The SSP UniTray® method is based on sequence specific primer amplification methods (SSP)
previously published. 1-3 The primer sets amplify the alleles described by the international
nomenclature committee of WHO.1 For details, see the Worksheet, Primer Mix Specificity Table
and Ambiguity List provided with each kit. The method has been extensively tested with
reference DNA from the International Workshops; UCLA Reference DNA Panel samples and other
well-characterized, serotyped samples.
*Taq DNA Polymerase, Ampli-Taq and the Gene-Amp PCR process are subject of patents and
patent applications of Hoffmann-LaRoche, USA.
In this manual:
Description
Section
Page
1.
Kit Components
2
2.
Material, Reagents, and Equipment not Supplied
2
3.
Sample Requirements
3
®
4.
Thermal Cycler SSP UniTray Amplification Profile
4
5.
Sample Setup Protocol
5
6.
Gel Electrophoresis
7
7.
Interpretation
8
8.
Limitations and Precautions
10
9.
Troubleshooting
11
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For In Vitro Diagnostic Use
1
Kit Components:
Description
1.1
96 well polycarbonate PCR trays
containing 5 µl/well of optimized
primer solution overlaid with
paraffin oil
1.2
75-580 µl aliquots of optimized
PCR Buffer containing dNTPs and
Gel Loading Buffer
1.3
2
Plastic sealers for sealing PCR
trays
Quantity
Storage
2 to 10 trays (kit
dependent)
-20° C in a NONFROST FREE
FREEZER
10-40 vials
(kit dependent)
-20° C in a NONFROST FREE
FREEZER
3 – 11 (kit
dependent)
Room temperature
10-40 (kit
dependent)
---------
11-41
(kit dependent)
---------
1
---------
1.4
Gel Documentation Form
1.5
Worksheets
1.6
Primer Mix Specificity Table
1.7
Certificate of Analysis/Tray
Configuration
1
1.8
Taq Polymerase, 5 units/µl
(optional)
Kit dependent
---------20° C in a NONFROST FREE
FREEZER
Material, Reagents, and Equipment not Supplied:
2.1
Taq DNA Polymerase, 5 units/µl:
2.1.1 The following enzymes are validated for use with the SSP UniTray® products:
Invitrogen™ Recombinant Taq DNA Polymerase; Roche Molecular
Systems, Taq DNA Polymerase; Perkin Elmer, Ampli-Taq DNA Polymerase;
Fisher, Taq DNA Polymerase; Advanced Biotechnologies Ltd., Taq DNA
Polymerase. Use of other DNA polymerase enzymes must be validated by the
user.
2.1.2 For the purchase of kits with Taq Polymerase and License fees, please refer
to the Invitrogen™ Product Catalog, or contact your Sales Representative.
See the last page of this manual for an important notice regarding kits sold
with Taq Polymerase.
2.2
Sterile, molecular grade water
2.3
Pipettors and tips: 1-10 µl
10-200 µl
100-1000 µl
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For In Vitro Diagnostic Use
2.4
Dispensing electronic pipettors: 100-250 µl capacity, capable of dispensing 8 µl
aliquots
2.5
8 channel pipettor: 5-25 µl adjustable volume
2.6
96 well thermal cycler with heated lid:
e.g., MJ Research, PTC-100 Peltier, PTC-200 DNA Engine, PTC-225 DNA Engine Tetrad;
Perkin Elmer, Gene Amp 9600; Perkin Elmer, Gene Amp 9700; and the LabLine,
Thermal Block II Model 212.
Note: Kits have been tested with the above thermal cyclers. Use of different
equipment will require user validation of thermal cycling parameters.
2.7
Invitrogen™ Heat Equalizing Block, Product Code # 900001D
2.8
TBE electrophoresis buffer (at 0.5X concentration)
2.9
DNA Molecular Weight markers to cover range of 50 – 2000 bp
2.9.1 Invitrogen™ PCR Markers, Product Code # 74601250 (recommended)
2.10
Invitrogen™ DNA Grade Agarose, Product Code # 75000500 (recommended)
2.11
Ethidium bromide (10 mg/ml)
Caution: Ethidium bromide is a mutagen. Handle with appropriate
personal protective equipment.
2.12
Electrophoresis Power supply
2.13
Recommended Electrophoresis System:
2.13.1 Electro-Fast® Electrophoresis System: 96 sample lanes plus separate marker
loading lanes.
This product can be purchased through Invitrogen™ Product code #920001.
2.13.2 Owl Centipede™ Extra Wide Minigel System, Model #: D3-14 with 4 Microtiter
combs, 25 teeth, 1.5 mm thick.
This product can be purchased through Invitrogen™ Product Code #800001
2.14
Recommended Gel Documentation System:
2.14.1 UV transilluminator
2.14.2 Polaroid camera with hood and filter for gel documentation
2.14.3 Polaroid film type 667
3
Sample Requirements:
3.1
DNA Sample in TE buffer or sterile water
1.05-10 µg total DNA for one full typing (kit dependent), equivalent to 14-80 µl (kit
dependent) at 75-125 ng/µl of DNA (see Table 5.0 at Sample Setup Section 5).
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For In Vitro Diagnostic Use
3.2
DNA isolated from blood samples should be collected in EDTA or ACD anticoagulated
tubes. DO NOT USE HEPARINIZED SAMPLES. Heparin may inhibit DNA
amplification.
3.3
Good quality DNA is critical to achieve an optimal result with the SSP UniTray®
System. Good quality DNA means that:
3.3.1 The OD260/280 is between 1.7 and 1.9 on diluted sample measured by UV
spectrophotometry
3.3.2 When checked on agarose gel electrophoresis, the major portion of DNA runs
slower than the 9.4 kb band of Hind III digested Lambda DNA marker
Note: DNA isolation may be performed by any qualified protocol that
produces high purity and good quality DNA. The Invitrogen™ DNA
Isolation Kit (Product Code #761001) and QIAamp® System are validated
for this use.
4
Thermal Cycler: SSP UniTray® Amplification Profile:
4.1
It is important to obtain rapid ramp times (~1° per second) and precise temperature
control for optimal results.
4.1.1 If using the Perking Elmer PE9700 thermal cycler, set ramping speed to the
Perkin Elmer PE9600 setting.
4.1.2 If using the MJ Research thermal cyclers, select the temperature control to
calculated, not block.
4.2
The following thermal cycler profile is optimized and validated for use with the
UniTray® SSP product line (see Section 2.6):
Step 1
1 minute at 96º C
Step 2 5 cycles of
96º C 25 seconds
70º C 50 seconds
72º C 45 seconds
Step 3 21 cycles of
96º C 25 seconds
65º C 50 seconds
72º C 45 seconds
Step 4 4 cycles of
96º C 25 seconds
55º C 60 seconds
72º C 120 seconds
Hold
4º C specify time
Total reaction volume (reaction + paraffin oil overlay) in each well = 23 µl
Note: The UniTray® is designed to be placed directly in the thermal cycler unit.
Do not use a tray holder or tray retainer.
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For In Vitro Diagnostic Use
5
Sample Setup:
Note: PCR buffer is aliquotted in single test volumes. Use one vial per test.
Use the following reference table (Table 5.0) to aid in PCR setup:
TABLE 5.0
Number of
Reactions
Per Typing
2-8 wells
9-12 wells
13-16 wells
17-24 wells
25-32 wells
33-40 wells
41-44 wells
45-48 wells
96 wells
5.1
Vol. of
Aliquotted
PCR buffer
(µl)
75.0 (1 vial)
85.0 (1 vial)
120.0 (1 vial)
150.0 (1 vial)
200.0 (1 vial)
250.0 (1 vial)
275.0 (1 vial)
300.0 (1 vial)
580.0 (1 vial)
Taq DNA
Polymerase
(µl)
Water
(µl)
1.2
1.4
1.9
2.4
3.2
4.0
4.4
4.8
9.3
30.0
34.0
48.0
60.0
80.0
100.0
110.0
120.0
268.0
Vol. of DNA
(75-125 ng/µl)
Added to Mix
(µl)
14.0
15.0
20.0
25.0
34.0
42.0
46.0
50.0
80.0
Final Vol. of
DNA and
Water (µl)
44.0
49.0
68.0
85.0
114.0
142.0
156.0
170.0
348.0
Water Control Tube.
5.1.1 Many laboratory accreditation standards require that a setup contamination
control reaction (Water Control Tube) be performed with each typing.
5.1.2 Add 50 µl of molecular reagent grade water to a clean 0.5 ml (or larger)
polypropylene tube.
5.1.3 Place the OPENED tube containing the 50 µl of water to one side of the test
setup area and proceed with test setup.
5.2
5.3
Thaw frozen PCR buffer (one vial for each test).
Remove one tray from freezer.
5.3.1 Record Tray Identification Number indicated on pouch label on the Gel
Documentation Form.
5.3.2 Carefully remove the SSP UniTray® from its pouch.
5.3.3 If only a single typing is to be run, use scissors to cut the UniTray® between
columns of adjacent tests (See product table Certificate of Analysis for
specific UniTray® primer mix layout). Return remaining tests to pouch. Store
at original conditions. Use within one month.
Note: When cutting a tray, cut from right to left, avoiding cutting off the
letters (A-H) in the leftmost column which aid in determining proper
UniTray® orientation.
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5.3.4 Do not allow paraffin oil to thaw before cutting or removing seal as this may
cause mix dispersal.
5.3.5 Place the tray inside a sample holder, such as a clear microtiter plate or a
0.2 ml tube rack (8 x 12).
5.3.6 Carefully remove adhesive seal from SSP UniTray®.
5.4
Remove Taq DNA Polymerase from freezer and keep chilled during setup (e.g., on
ice).
5.5
Add water and Taq DNA Polymerase to the PCR buffer (from section 5.2) and mix
thoroughly. (See Table 5.0 for appropriate volumes of water and Taq).
5.6
Note: Thaw tray completely before adding PCR buffer mixture. Remove 7 µl
from this mixture (Section 5.5) and add to the contamination control well of the
individual typing. The contamination control well is the last well of each primer mix
set. See Kit Tray Configuration for exact well position(s).
5.7
Add 1µl of water from the water control tube (see Section 5.1) to the contamination
control well referenced in Section 5.6 above.
5.8
Add the volume of completely dissolved DNA sample (75-125 ng/µl) to the
remaining buffer mixture, as indicated by Table 5.0, and mix thoroughly.
5.9
Using an electronic dispensing pipettor, dispense 8 µl into each of the remaining
wells. Be careful to dispense the drops onto the side walls of the wells, near each
well’s top, allowing the dispensed drop to slide under the paraffin oil. Do not allow
the pipette tip to come in contact with the well contents.
Note: Confirm that each well contains sample by noting the color of the solution
in each well. A settled sample will be indicated by a purple solution color. If a
drop is hung up on the side of a well, GENTLY tap the tray in the holder against
the bench top to ensure proper mixing of the DNA sample and primer.
Sufficient volume is supplied to allow for pipetting losses.
5.10
Remove backing from an adhesive plastic seal and place over the top of the tray.
5.10.1 Gently press the seal onto the tray, making sure that the tray is completely
sealed.
5.10.2 Trim the edges of the plastic seal, if necessary. It is normal for the plastic
seal above the wells to appear indented upon completion of the thermal
cycling run. This does not affect amplification in any way.
5.11
Set the tray in thermal cycler and place the Heat Equalizing Block on top of the
sealed tray. Close the lid and tighten. Begin thermal cycling.
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For In Vitro Diagnostic Use
Note: It is very important that the sealed tray be seated firmly in the thermal
cycler so that all wells are in contact with the block. It is necessary to place the
Heat Equalizing Block on top of the sealed plate to ensure firm contact and heat
transfer. This is required even when using a heated lid. Do not use the sample
holder supplied by Perkin Elmer with the SSP UniTray®. See Troubleshooting
(Section 9.4): Overall poor or absent amplification.
Note: Sections 5.9 through 5.11 must be performed efficiently to minimize time
between sample addition and initiation of thermal cycling. Prolonged
incubation at room temperature (greater than 5 minutes) may cause
mispriming and nonspecific PCR reactions.
5.12
6
After thermal cycling, remove tray and proceed to gel electrophoresis. If not
performing electrophoresis immediately, store tray at 4º C for up to one week.
Gel Electrophoresis:
General Directions: Use a high quality agarose, capable of resolving 50-2000 base pair
fragments of DNA. Invitrogen™ DNA Grade Agarose (Product Code #75000500) works well at
2%. Prepare the gel in 0.5X TBE buffer. After cooling to 60º C add 2 µl of 10 mg/ml ethidium
bromide for each 100 ml agarose solution and mix well. Pour into casting tray and allow to cool
for at least 30 minutes. Use 0.5X TBE buffer in gel chamber as a running buffer. Gels can be
run at 10 volts per centimeter gel length.
The following directions are for the Owl Centipede™, Extra Wide Minigel System only. Other gel
systems will require different amounts of agarose and electrophoresis conditions. Consult your
specific equipment protocol for assistance, or contact technical support personnel at Invitrogen™.
If using the Electro-Fast® Electrophoresis System, follow the instructions for
electrophoresis included with these units.
6.1
Pour a 160 ml 2% agarose gel following the guidelines above. Use four 25 well
microtiter format combs and place one at the top and the others at equal distances
below.
6.2
Fill electrophoresis chamber with 0.5X TBE buffer.
6.3
Carefully remove seal from UniTray®.
6.3.1 Holding the tray firmly inside a holder, carefully fold back the seal from one
edge.
6.3.2 Caution: Sudden movement of the tray can disperse amplified product and
oil, contaminating the laboratory and may require repetition of the test.
Note: It is recommended that the tray be cooled at 4º C for five minutes
to let the paraffin oil solidify before the seal is removed. This will help
prevent accidental dispersal of amplified product during seal removal.
6.4
Load 5 µl PCR marker to the appropriate lane(s) of the gel (See Gel
Documentation Form).
If using the Electro-Fast®, carefully load 2 µl of the PCR marker into the designated
wells.
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For In Vitro Diagnostic Use
6.5
Using an 8 channel pipettor, carefully transfer 8 µl of PCR products/gel loading
buffer from the tray, begin with wells A-1 through H-1, to the gel lanes (See Gel
Loading Template 6.5).
If using the Electro-Fast®, carefully load 6 µl of the PCR product into the wells.
Note: Be certain to keep the tips at the bottom of the well while slowly drawing
up sample into the pipette tips. With a paper towel, blot off any oil remaining
in the tips. Gently load the samples into the gel.
Gel Loading Template 6.5
1
A
lane 1
B
lane 2
C
D
E
2
3
4
5
6
7
8
9
10
11
12
See Certificate of Analysis for Kit
Specific Tray Configuration
F
G
H
Note: The word “well” refers to the tray location assignment, while the word
“lane” refers to a well’s corresponding gel lane.
6.6
Electrophoretically separate the DNA at 150 volts for 18-23 minutes using the Owl
Centipede™ unit, or until the orange dye front in the Invitrogen™ PCR Marker
approaches the next row of wells.
Note: The purple sample dye will be at approximately 300 bp after running the
gel for 18-23 minutes.
6.7
Turn off power, disconnect electrodes and remove gel. Photograph gel over UVtransilluminator.
Note: Use of the Invitrogen™ Fluorescent Numbering Panel may aid in positive
lane identification. Order the Centipede™ system Numbering Panel by
specifying Product Code # 810011. Order the Electro-Fast® system Numbering
Panel by specifying Product Code # 920011.
Note: To aid in troubleshooting and technical support it is helpful for Invitrogen
Corporation to obtain an original gel photo. For this purpose, the user may
want to take additional photos.
7
Interpretation:
7.1
Affix the gel photo to the Gel Documentation Form.
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For In Vitro Diagnostic Use
7.1.1 On the Gel Documentation Form, “M” refers to the marker lane.
7.2
Examine gel photo carefully and determine the positive lanes.
7.2.1 Each lane of the gel, containing a loaded sample, should show a control band
except the lane which contains the contamination control well. See the Gel
Documentation Form for details on the internal control sizes.
7.2.2 The control band may or may not amplify efficiently when there is specific
product present due to substrate competition. The control primers are
present in lower concentration in order to favor the allele specific reaction.
7.2.3 Weak bands above the internal control (except the 200 base pair internal
control) may appear in all lanes. These are not of particular concern. They
are an indication of robust amplification.
7.2.4 Absent control bands with no specific amplification are indicative of failed
reactions.
7.2.4.1
If alleles can be determined in the presence of a failed PCR reaction,
and that failed reaction does not change the allele assignment, the
test does not need to be repeated.
7.2.4.2
If, however, there is an apparent homozygous result, or the missed
reaction could change an allele assignment, the typing must be
repeated.
7.2.5 If weak bands of incorrect product size are present, disregard them if the
overall strength and clarity of the amplification is good.
7.2.6 Unused primers will form a diffuse band below 50 base pairs.
7.2.7 Primer dimer usually appears above the primer band, but below the area
where specific product is found; this appears as a fuzzy band below 80 base
pairs.
7.2.8 Several lanes have two or more possible sizes of PCR products. These wells
have multiplexed primer pairs which give rise to different, amplicons
depending upon the allele present. Refer to the Primer Mix Specificity Table
for further information for allele assignment.
7.2.9 See Primer Mix Specificity Table and ambiguity list provided with each kit for
details on resolution.
7.2.10 False negative reactions can be caused by inefficient amplification, poor
quality of DNA, uneven placement of the plate in the block, temperature
variations across the wells of the thermal cycler itself, or inadequate thermal
cycler calibration.
7.2.10.1
False negative reactions rarely occur when the control band is
present
7.2.10.2
It is possible that the false negatives are due to a new or yet
uncharacterized allele.
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For In Vitro Diagnostic Use
7.2.11 The contamination control well contains primer pairs that amplify DNA
produced by either specific PCR amplifications or genomic DNA.
8
7.2.11.1
If the negative buffer/Taq mixture was added as directed, any band
in this lane is evidence of contamination and the results of the test
are invalid.
7.2.11.2
A primer dimer band of <80 base pairs may be present. This does
not invalidate the test. Primer dimers are known to occur
occasionally.
7.3
Confirm the approximate product size using the Gel Documentation Form or
Worksheet.
7.4
Mark the positive lanes on the worksheet.
7.5
Using a highlighting pen, highlight each positive lane (column) with a vertical line
running through the chart columns from top to bottom.
7.6
Align a ruler across the first row of the worksheet and scan from the left to right.
Examine each row from top to bottom, making the first allele assignment only
where all the black boxes for that allele choice fall within the highlighted lanes.
7.7
Assign the second allele choice if needed by continuing to scan the remaining alleles
as above. Be sure to complete an entire review of the worksheet to determine all
possible allele assignments. You must account for all positive lanes at least
once. However, lanes can be used to assign more than one allele.
7.8
Be certain that all the lanes required for a particular allele are positive before
making a specific allele assignment. Refer to Section 9 for reaction patterns that do
not give a typing result.
7.9
For High Resolution SSP UniTray®, assignments should not be made for allele
groups exhibiting positive reactions other than the groups for which the test was
designed, i.e., B*15 assignments should not be made from a B*35 High Resolution
SSP UniTray®.
Limitations and Precautions:
8.1
Before implementing the SSP UniTray® method in your laboratory, perform quality
assurance and quality control for amplification based methods using known
molecularly typed samples. Such samples can be obtained from the International
Workshop Reference Cell Panel and the UCLA DNA Reference panel. Be sure to
consult the Primer Mix Specificity Table and Ambiguity List for details on
coverage.
8.2
Every attempt has been made to validate all primer mixes used in this kit with
molecularly typed DNA samples or well-characterized serologically typed samples.
Due to the lack of access to molecularly typed reference material, some wells may
not have been validated using positive control DNA. Refer to the Certificate of
Analysis for detailed information.
8.3
HLA typing using the Invitrogen™ SSP UniTray® must be performed in the presence
of a qualified Director, Technical Supervisor and/or general Supervisor following
accepted laboratory accreditation standards. We must emphasize that these
products are for professional use only.
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For In Vitro Diagnostic Use
9
Troubleshooting:
General Problems
9.1
Use of excess sample DNA (200 ng/µl or more per reaction) may favor nonspecific
PCR products. An intense smear of high molecular weight DNA present on gel
photos of amplified products may indicate that excess DNA was used. A general
weak amplification might indicate that less than the required amount of sample DNA
was used in the reaction (<75 ng/µl per reaction) which may cause false negative
reactions.
9.2
RNA contamination may cause overestimation of DNA concentration when measured
by spectrophotometry. A way to verify the reading is to run a small aliquot of
sample DNA (about 200 ng) in a 0.7% agarose gel and compare it with a DNA
marker of known concentration.
9.3
Degraded DNA may not amplify reliably with UniTray®. Verify DNA integrity as
above. Obtain another sample and repeat the DNA extraction. For technical details,
please contact Invitrogen Corporation.
9.4
Problem: Overall poor or absent amplification indicated by weak control bands,
absent or negative allele specific bands.
(Possible Causes)
9.4.1 Inadequate contact between thermal cycler block and tray – DO NOT use the
tray holder or tray retainer provided by Perkin Elmer with the UniTray®.
9.4.2 Heparinized samples – use EDTA or ACD as anticoagulants.
9.4.3 Poor quality DNA – when using the Invitrogen™ DNA Isolation Kit, clean up
DNA by repeating protease digestion, or re-purify the DNA sample. As a last
resort, extract a fresh sample.
9.4.4 Low DNA concentrations –use more DNA and adjust the water volume
accordingly to buffer mixture or concentrate DNA.
9.4.5 Inhibitors present – make sure that DNA is of good quality (see section 3.3)
before setting up PCR.
9.4.6 Degraded DNA sample – is apparent by presence of a smear in the gel lanes.
Isolate DNA from a fresh sample.
9.4.7 Improperly calibrated thermal cycler – recalibrate thermal cycler.
9.4.8 Lack of Taq DNA Polymerase activity – verify activity of Taq with a known
reference DNA sample.
9.4.9 Annealing temperature not optimal – decrease the annealing temperature in
step 2 of the amplification profile from 70º C to 69º C and in step 3 of the
amplification profile from 65º C to 64º C (Section 4.2).
9.5
Problem: Random failures: more than 1 failed lane
(Possible Causes)
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For In Vitro Diagnostic Use
9.5.1 DNA is not evenly re-suspended in diluent – pipet DNA up and down several
times to aid mixing; alternatively, heat DNA on a heat block 10 minutes at
70º C to dissolve.
9.5.2 DNA not mixed adequately with PCR buffer –mix thoroughly before adding to
tray.
9.5.3 Uneven volume of buffer/Taq/DNA solution added – exercise caution when
dispensing samples. Make sure all of the reaction mixture is contained under
the paraffin oil.
9.5.4 Inadequate contact between thermal cycler block and tray – DO NOT use the
tray holder or tray retainer provided by Perkin Elmer with UniTray®.
9.6
Problem: False positives
(Possible Causes)
9.6.1 Excess DNA or Taq Polymerase - measure DNA with UV spectrophotometry.
9.6.2 Extensive delay between PCR setup and start of thermal cycling – no more
than a 5 minute delay should be allowed before thermal cycling.
9.6.3 Incorrect order in gel loading – check alignment of mixes and gel lanes.
9.6.4 Interpretation of primer dimer as specific bands – check correct band size.
9.7
Problem: False negatives
(Possible Causes)
9.7.1 Improperly calibrated thermal cycler – recalibrate thermal cycler.
9.7.2 If recalibration does not correct the problem, re-test the sample with a
previously typed reference sample with the same allelic specificity. If
confirmed as negative, call Invitrogen Corporation for technical
support.
9.7.3 Failure of buffer/Taq/DNA drop to pass through paraffin oil. Briefly centrifuge
tray before thermal cycling or tap gently on bench top.
9.7.4 Incorrect order in gel loading – check alignment of mixes and gel lanes.
9.7.5 Annealing temperature not optimal – decrease the annealing temperature
instep 2 of the amplification profile from 70º C to 69º C and in step 3 of the
amplification profile from 65º C to 64º C.
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9.8
Problem: Overall fuzzy bands, smeared lanes
(Possible Causes)
9.8.1 Gel is too thin due to excess evaporation while heating – compensate for lost
volume by adding water.
9.8.2 Agarose not completely dissolved – boil for an additional 30 seconds after
melting.
9.8.3 Overheating gel, too high voltage – lower voltage.
9.8.4 TBE concentration too high – concentration should be 0.5X TBE.
9.8.5 Heavy streaking in random wells can be caused by uneven suspensions of
DNA – using an 8 channel pipettor, mix the PCR product up and down two
times before loading.
9.8.6 Rapid release of amplified product during gel loading can cause product to
float out of well – use slow, steady pipetting when loading gel.
9.9
Problem: Gel picture too dark
(Possible Causes)
9.9.1 Forgot to add ethidium bromide, or added the wrong amount – use 2 µl
ethidium bromide (10 mg/ml) for each 100 ml agarose solution.
9.9.2 Gel tray not UV transparent – remove gel from tray before viewing.
9.9.3 Incorrect camera setting – increase exposure time or aperture setting.
9.10
Problem: Gel picture too bright
(Possible Causes)
9.10.1 Excess amount of ethidium bromide – use 2 µl ethidium bromide for each
100ml agarose solution.
9.10.2 Incorrect camera setting – decrease exposure time or aperture setting.
9.11
Problem: Occasional faint lanes
(Possible Causes)
9.11.1 Product floated out of well – pipette tips need to be properly aligned with gel
wells.
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For In Vitro Diagnostic Use
Disclaimer:
NOTICE TO PURCHASER: SSP UniTray® Kits without Taq Polymerase – This product is
optimized for use in the Polymerase Chain Reaction (“PCR”) Process which is covered by patents
by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. (“Roche”). No license under
these patents to use the PCR Process conveyed expressly or by implication to the purchaser by
the purchase of this product. Further information on purchasing licenses to practice the PCR
Process may be obtained by contacting the Director of Licensing at Roche Molecular Systems,
Inc., 1145 Atlantic Avenue, Alameda, CA 94501.
NOTICE TO PURCHASER: SSP UniTray® Kits with Taq Polymerase – The purchase price of this
product includes limited, non-transferable rights under U.S. Patents 4,683,202, 4,683,195 and
4,965,188 and their foreign counterparts, owned by Roche Molecular Systems, Inc. and F.
Hoffmann-La Roche Ltd. (“Roche”) to use only this amount of product to practice the Polymerase
Chain Reaction (“PCR”) Process described in said patents solely for HLA Typing applications of
the purchaser solely for organ or tissue or bone marrow transplantation, and explicitly excludes
analysis of forensic evidence or parentage determination. The right to use this product to
perform and to offer commercial services for HLA typing for organ or tissue transplantation using
PCR, including reporting the results of the purchaser’s activities for a fee or other commercial
consideration, is also hereby granted. Further information on purchasing licenses to practice the
PCR Process may be obtained by contacting, in the United States, the Director of Licensing at
Roche Molecular Systems, Inc. 1145 Atlantic Avenue, Alameda, CA 94501, and outside the
United States, the PCR Licensing Manager, F. Hoffmann-La Roche Ltd., Grenzacherstr. 124, CH4070 Basel, Switzerland.
®
The Invitrogen™ UniTray Product Line uses ARMS™ technology and is sold under license from
ZENECA Limited. ARMS is the subject of European Patent No. 0332435, US Patent No. 5595890
and corresponding worldwide patents. ARMS is a trademark of ZENECA Limited.
References:
1. Current HLA alleles can be found at: http://www.anthonynolan.org.uk/HIG/index.html
2. Bunce M., O’Neil C., Barnardo M., Morris P., Welsh K. Phototyping: Comprehensive DNA
typing for HLA-A, B, C, DRB 3, DRB 4, DRB 5 and DQB 1 by PCR with 144 primer mixes
utilizing sequence-specific primers (PCR-SSP) Tissue Antigens V 46 November 1995.
3. Olerup, O. and Zetterquist, H. HLA-DR typing by PCR amplification with sequence specific
primer (PCR-SSP) In 2 hours: An alternative to serological DR typing in clinical practice
including donor-recipient matching in cadaveric transplantations. Tissue Antigens V. 39:
225-235, 1992
PR001
Revision 11
Print 9/06
14
For In Vitro Diagnostic Use
European Representative:
Invitrogen Ltd.
11 Bassendale Road
Croft Business Park
Bromborough, Wirral
CH62 3QL, U.K.
Tel: 44 151 346 1234
Invitrogen Corporation
9099 North Deerbrook Trail
Brown Deer, Wisconsin 53223 USA
Tel: (800) 955-6288
Fax: (800) 331-2286
www.invitrogen.com
Self-Declared Products (CE marked)
451404
451414
451606D
451616D
451703
457173
4719010
4719110
7830010
7830110
DQB1 SSP UniTray Kit - for HLA tissue typing
DQB1 SSP UniTray Kit with Taq Polymerase - for HLA tissue typing
DPB1 SSP UniTray Kit - for HLA tissue typing
DPB1 SSP UniTray Kit with Taq Polymerase - for HLA tissue typing
DQA1 SSP UniTray Kit - for HLA tissue typing
DQA1 SSP UniTray Kit with Taq Polymerase - for HLA tissue typing
Cw High Res SSP UniTray Kit - for HLA tissue typing
Cw High Res SSP UniTray Kit with Taq Polymerase - for HLA tissue
typing
C LOCUS SSP UniTray Kit - for HLA tissue typing
C LOCUS SSP UniTray Kit with Taq Polymerase - for HLA tissue typing
15
For In Vitro Diagnostic Use
For country-specific contact information visit our website at
www.invitrogen.com
Invitrogen Corporation
9099 N. Deerbrook Trail
Brown Deer, WI 53223 USA
Tel. 1-800-955-6288
Fax 1-800-331-2286
Email: [email protected]
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