Thermo Fisher Scientific GlobalFiler User Manual
GlobalFiler™ Express PCR Amplification Kit
USER GUIDE
Catalog Numbers 4476609 and 4474665
Publication Number 4477672
Revision G
For Research, Forensic, or Paternity Use
Only. Not for use in diagnostic procedures.
Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. 4477672
Revision Date Description
G 13 October 2020 Add the SeqStudio™ Genetic Analyzer. Consolidate sample preparation for
F 09 June 2020 In kit overview, change amplification time from ~80 to ~45 minutes. Update
E 21 December 2016 Revised the Peak Detector tab settings for GeneMapper™ ID‑X Software analysis.
D 06 October 2016 Updated 3730 Peak Detector settings in Chapter 4. Add references to 3500 Series
C May 2014 Added data to Chapter 5 about the evaluation of Hardy-Weinberg equilibrium.
B April 2014 Added Master Mix Additive instructions. Updated the HID Updater 3500 DC v2.0
A October 2012 New document
electrophoresis; it is the same for all instruments.
copyright page to latest template. On cover, update regulatory statement and
remove licensing statement link.
Data Collection 3 and GeneMapper ID-X v1.5.
Non-technical changes: Reorganized Chapter 1 and Chapter 5.
instructions, including sizing method information. Added Chapter 5, Experiments
and Results.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Windows
and Windows Vista are trademarks of Microsoft Corporation. EasiCollect , Whatman, and FTA are trademarks of Whatman Limited.
NUCLEIC-CARD, FLOQ,Swabs, and Copan are trademarks of Copan Flock Technologies, and used by Thermo Fisher Scientific under
their permission. Bode Buccal DNA Collector is a trademark of Bode Technology Group, Inc. Harris Micro-Punch is a trademark
of Harris, Joel S. TA Shunderson Communications. VWR Scientific is a trademark of VWR International, Inc. Robbins Scientific is
a trademark of Molecular Bioproducts, Inc. Agilent is a trademark of Agilent Technologies, Inc. Adobe, Acrobat, and Reader are
trademarks of Adobe Systems Incorporated.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 8
Product description ............................................................. 8
Kit overview ................................................................ 8
Single-source sample types supported ........................................ 9
Substrate examples ......................................................... 9
About the primers ........................................................... 9
Dyes used in the kit ........................................................ 10
Loci amplified by the kit .................................................... 10
Standards and controls that are required ..................................... 11
Allelic ladder profile ........................................................ 12
DNA Control 007 profile .................................................... 13
Contents and storage ........................................................... 14
Required materials not supplied ................................................. 15
Instrument and software compatibility ............................................ 15
Workflow ..................................................................... 17
■
CHAPTER 2 Perform PCR ....................................................... 18
Optimize PCR cycle number (before first use of the kit) ............................. 18
Procedural guidelines when optimizing PCR cycle number ...................... 18
Select samples and prepare plates ........................................... 18
Determine optimum PCR conditions ......................................... 19
Before you begin ............................................................... 19
Thaw reagents and prepare Master Mix (before first use of the kit) ............... 19
Treated paper substrates: prepare the amplification kit reactions ..................... 20
Sample preparation guidelines: treated paper substrate ........................ 20
Prepare low-TE buer ...................................................... 20
Prepare the amplification kit reactions: treated paper substrate .................. 21
Untreated paper substrates: prepare the amplification kit reactions ................... 22
Sample preparation guidelines: untreated paper substrate ...................... 22
Prepare the amplification kit reactions: untreated paper substrate ................ 23
Swab substrates: prepare the amplification kit reactions ............................ 24
Sample preparation guidelines: swab substrate ................................ 24
Prepare the sample lysate: room temperature ................................. 25
Prepare the sample lysate: heat protocol ..................................... 25
GlobalFiler
™
Express PCR Amplification Kit User Guide
3
Contents
■
Prepare the reactions: swab substrate ........................................ 26
Store the sample lysate ..................................................... 27
Perform PCR .................................................................. 28
CHAPTER 3 Perform electrophoresis ............................................ 29
Allelic ladder requirements for electrophoresis ..................................... 29
Materials required for electrophoresis ............................................. 30
Set up the SeqStudio™ instruments for electrophoresis (before first use of the kit) ...... 30
Electrophoresis software setup .............................................. 30
Perform spectral calibration ................................................. 31
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) ..... 32
Electrophoresis software setup .............................................. 32
Obtain and run the HID Updater (v1 and v2 software only) ...................... 33
Modify 3500 QC protocol ................................................... 33
Perform spectral calibration ................................................. 35
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) ...... 36
Electrophoresis software setup .............................................. 36
Obtain and activate 6-dye license ............................................ 36
Perform spectral calibration ................................................. 38
Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit) ...... 38
Electrophoresis software setup .............................................. 38
Obtain and activate the 6-dye license ........................................ 39
Perform spectral calibration ................................................. 40
Prepare samples for electrophoresis .............................................. 41
■
CHAPTER 4 Analyze data with GeneMapper™ ID‑X Software ................ 42
Overview of GeneMapper™ ID‑X Software ......................................... 42
Allelic ladder requirements for data analysis ....................................... 43
File names and versions used in this section ...................................... 43
Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit) ........ 44
Workflow: Set up GeneMapper™ ID‑X Software ............................... 44
Check panel, bin, and stutter file versions on your computer .................... 44
(If needed) Download newer versions of panel, bin, and stutter files .............. 45
Import panels, bins, and marker stutter ....................................... 45
(Optional) Define custom table or plot settings ................................. 48
Create an analysis method ...................................................... 49
Create an analysis method .................................................. 49
Enter Analysis Method settings .............................................. 50
Create a size standard definition file if needed ..................................... 57
About the GS600_LIZ_ (60– 460) size standard definition file .................... 57
If you use POP-7™ polymer on a 3730 instrument .............................. 57
Create a size standard definition file .......................................... 58
Analyze and edit sample files with GeneMapper™ ID‑X Software ..................... 60
4
GlobalFiler™ Express PCR Amplification Kit User Guide
Examine or edit a project ........................................................ 61
For more information on using the GeneMapper™ ID‑X Software ..................... 61
■
CHAPTER 5 Experiments and results ........................................... 62
Importance of validation ........................................................ 62
Experiment conditions .......................................................... 62
Laboratory requirements for internal validation ..................................... 63
Developmental validation ....................................................... 63
SWGDAM guideline 2.2.1 ................................................... 63
SWGDAM guideline 3.9.2 ................................................... 63
PCR components .......................................................... 63
PCR cycle number ......................................................... 65
Thermal cycling temperatures ............................................... 65
Accuracy, precision, and reproducibility ........................................... 66
SWGDAM guideline 3.5 ..................................................... 66
Accuracy observation ...................................................... 67
Precision and size window description ....................................... 69
Precision observation ...................................................... 70
Extra peaks in the electropherogram .............................................. 91
Causes of extra peaks ...................................................... 91
Extra peaks: Stutter ........................................................ 91
Extra peaks: Addition of 3' A nucleotide ...................................... 99
Extra peaks: Artifacts ..................................................... 101
Characterization of loci ........................................................ 102
SWGDAM guideline 3.1 ................................................... 102
Loci in this kit ............................................................ 102
Nature of polymorphisms ................................................. 102
Inheritance ............................................................... 102
Mapping ................................................................. 102
Genetic linkage ........................................................... 103
Species specificity ............................................................ 103
SWGDAM Guideline 3.2 ................................................... 103
Nonhuman study observation .............................................. 104
Sensitivity .................................................................... 105
SWGDAM guideline 3.3 ................................................... 105
Sample collection factors that can aect DNA quantity ........................ 105
Eect of DNA quantity on results ........................................... 105
Sensitivity observation .................................................... 107
Contents
GlobalFiler™ Express PCR Amplification Kit User Guide
5
Contents
■
■
Stability ...................................................................... 109
SWGDAM guideline 3.4 ................................................... 109
DNA on FTA™ cards ....................................................... 109
DNA on 4N6FLOQSwabs™ sample collectors ................................ 110
Population data ............................................................... 111
SWGDAM guideline 3.7 ................................................... 111
Population data overview .................................................. 111
Loci in the kit ............................................................. 112
Population samples used in these studies ................................... 112
Concordance studies ..................................................... 112
Probability of Identity definition ............................................. 112
Probability of identity observation ........................................... 113
Probability of paternity exclusion observation ................................ 131
APPENDIX A Troubleshooting .................................................. 135
APPENDIX B Materials required but not supplied ............................ 138
STR kit required materials ...................................................... 138
Sample preparation required materials ........................................... 138
Treated paper substrate ................................................... 138
Untreated paper substrate ................................................. 139
Swab substrate .......................................................... 139
Thermal cycler required materials ............................................... 140
Veriti™ Thermal Cycler ..................................................... 140
GeneAmp™ PCR System 9700 ............................................. 140
Genetic analyzer required materials ............................................. 141
SeqStudio™ Genetic Analyzer .............................................. 141
3500 Series Genetic Analyzer .............................................. 141
3130 Series Genetic Analyzer .............................................. 142
3730 Series Genetic Analyzer .............................................. 142
Analysis software required materials ............................................. 143
GeneMapper™ ID‑X Software ............................................... 143
Miscellaneous required materials ............................................... 143
Plates and tubes ......................................................... 143
Laboratory supplies ....................................................... 144
■
APPENDIX C Plate layouts ...................................................... 145
Example PCR plate layout ..................................................... 145
Example electrophoresis plate layout ............................................ 145
6
GlobalFiler™ Express PCR Amplification Kit User Guide
■
APPENDIX D PCR work areas .................................................. 146
Work area setup and lab design ................................................ 146
PCR setup work area materials ................................................. 146
Amplified DNA work area ...................................................... 147
■
APPENDIX E Safety .............................................................. 148
Chemical safety .............................................................. 149
Biological hazard safety ....................................................... 150
■
Documentation and support ...................................................... 151
Related documentation ........................................................ 151
Customer and technical support ................................................ 153
Limited product warranty ...................................................... 153
References
Contents
Index ..................................................................................... 158
GlobalFiler™ Express PCR Amplification Kit User Guide
7
1
Product description .................................................................... 8
■
Contents and storage ................................................................. 14
■
Required materials not supplied ........................................................ 15
■
Instrument and software compatibility ................................................... 15
■
Workflow ............................................................................ 17
■
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
Kit overview
Product information
The Applied Biosystems™ GlobalFiler™ Express PCR Amplification Kit is a 6-dye, short tandem repeat
(STR) multiplex assay for the amplification of human genomic DNA.
The kit amplifies:
•
21 autosomal STR loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51,
D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248,
D1S1656, D12S391, D2S1338)
•
1 Y-STR (DYS391)
•
1 insertion/deletion polymorphic marker on the Y chromosome (Y indel)
•
Amelogenin (sex determining marker)
The GlobalFiler™ Express PCR Amplification Kit combines the 13 original CODIS loci with 7 from the
expanded European Standard Set of Loci (ESSL) and the highly discriminating SE33 locus. The kit
delivers a 24-locus multiplex with the highest discrimination power of any Thermo Fisher Scientific
Human Identification Kit, along with high sensitivity and tolerance to inhibitors. The concentration of 10
mini-STR loci that are entirely below 220 bp maximizes performance on degraded samples. The highly
optimized buer formulation contains an enzyme that allows completion of amplification in ~45 minutes.
The GlobalFiler™ Express PCR Amplification Kit uses the same improved process for synthesis and
purification of the amplification primers developed for other next-generation Thermo Fisher Scientific
STR chemistries. The improved amplification primers deliver clean electrophoretic backgrounds that
assist interpretation.
8
GlobalFiler™ Express PCR Amplification Kit User Guide
Single-source sample types supported
The GlobalFiler™ Express PCR Amplification Kit is optimized to allow direct amplification from the
following types of single-source samples without the need for sample purification:
•
Blood and buccal samples on treated paper substrates.
•
Blood and buccal samples collected on untreated paper substrates and treated with Prep‑n‑Go
Buer.
•
Buccal samples collected on swab substrates and treated with Prep‑n‑Go™ Buer.
Substrate examples
•
Treated paper: NUCLEIC-CARD™ system or Whatman FTA™ cards
•
Untreated paper: Bode Buccal DNA Collector™ or 903 paper
•
Swab: FLOQSwabs™ or cotton swabs
Note: Our testing does not include blood samples on swab substrates. This sample type is not
typically used for the collection of reference samples.
Chapter 1
Product information
Product description
1
™
About the primers
The GlobalFiler™ Express PCR Amplification Kit primers are manufactured using the same synthesis
and purification improvements as the primers in the NGM SElect™ and the Identifiler™ Plus kits. These
improvements enhance the assay signal‑to‑noise ratio and simplify the interpretation of results.
The primers used in the kit are:
•
For all loci except AMEL—The same primer sequences as the NGM SElect™ kit and the Identifiler
Plus kit including SNP-specific primers for the vWA, D16S539, AMEL, D2S441, D22S1045, and
D8S1179 loci.
•
For AMEL—The same primer sequences as the NGM SElect™ kit (which are dierent from the
Identifiler™ Plus kit).
The GlobalFiler™ Express PCR Amplification Kit also includes the following primer additions and
modifications:
•
Addition of DYS391 and a novel Y indel.
•
The TPOX reverse primer has been redesigned to relocate the amplicon into the higher size range
of the multiplex and optimize marker spacing.
•
Addition of 8 new SNP-specific primers for the D3S1358, vWA, D18S51, D19S433, TH01, FGA,
D5S818, and SE33 loci. The second degenerate primer was added to the vWA locus to address
two dierent SNPs in the primer binding site.
Non-nucleotide linkers are used in primer synthesis for the following loci: D19S433, vWA, CSF1PO,
D2S441, TH01, FGA, and D12S391. For these primers, non-nucleotide linkers are placed between the
primers and the fluorescent dye during oligonucleotide synthesis (Butler 2005, Grossman et al., 1994).
Non-nucleotide linkers enable reproducible positioning of the alleles to facilitate interlocus spacing. The
combination of a 6-dye fluorescent system and the use of non-nucleotide linkers allows simultaneous
amplification and ecient separation of all 24 markers during automated DNA fragment analysis.
™
GlobalFiler™ Express PCR Amplification Kit User Guide
9
Chapter 1 Product information
1
Product description
Dyes used in the kit
Dye Color Label
™
6‑FAM
VIC
NED
TAZ
SID
LIZ
™
™
™
™
™
Blue Samples, allelic ladders, and controls
Green
Yellow
Red
Purple
Orange GeneScan™ 600 LIZ™ Size Standard v2.0
Loci amplified by the kit
Table 1 GlobalFiler™ Express PCR Amplification Kit loci and alleles
Locus
designation
D3S1358 3p21.31 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 6-FAM
vWA 12p13.31 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
Chromosome
location
Alleles included in Allelic Ladder Dye label
™
DNA Control
007
15, 16
14, 16
24
D16S539 16q24.1 5, 8, 9, 10, 11, 12,13, 14, 15 9, 10
CSF1PO 5q33.3-34 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 11, 12
TPOX 2p23-2per 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 8, 8
Y indel Yq11.221 1, 2 VIC
Amelogenin X: p22.1-22.3
X, Y X, Y
™
Y: p11.2
D8S1179 8q24.13 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 12, 13
D21S11 21q11.2-q21 24, 24.2, 25, 26, 27, 28, 28.2, 29, 29.2, 30, 30.2,
28, 31
31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2,
36, 37, 38
D18S51 18q21.33 7, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15, 16,
12, 15
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27
2
DYS391 Yq11.21 7, 8, 9, 10, 11, 12, 13 11
D2S441 2p14 8, 9, 10, 11, 11.3, 12, 13, 14, 15, 16, 17 NED
™
D19S433 19q12 6, 7, 8, 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15,
15.2, 16, 16.2, 17, 17.2, 18.2, 19.2
TH01 11p15.5 4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3 7, 9.3
10
GlobalFiler™ Express PCR Amplification Kit User Guide
14, 15
14, 15
Chapter 1
Table 1 GlobalFiler Express PCR Amplification Kit loci and alleles (continued)
Product information
Product description
1
Locus
designation
FGA NED
D22S1045 22q12.3 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 TAZ
D5S818 5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 11, 11
D13S317 13q22-31 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 11, 11
D7S820 7q11.21-22 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 7, 12
SE33 6q14 4.2, 6.3, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19,
D10S1248 10q26.3 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 SID
D1S1656 1q42.2 9, 10, 11, 12, 13, 14, 14.3, 15, 15.3, 16, 16.3, 17,
Chromosome
location
4q28 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 26.2, 27, 28, 29, 30, 30.2, 31.2, 32.2, 33.2,
42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2,
51.2
20, 20.2, 21, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2,
27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35,
35.2, 36, 37
17.3, 18.3, 19.3, 20.3
Alleles included in Allelic Ladder Dye label
DNA Control
007
™
™
™
24, 26
11, 16
17, 25.2
12, 15
13, 16
D12S391 12p13.2 14, 15, 16, 17, 18, 19, 19.3, 20, 21, 22, 23, 24,
25, 26, 27
D2S1338 2q35 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28
Standards and controls that are required
For the GlobalFiler™ Express PCR Amplification Kit, the panel of standards needed for PCR
amplification, PCR product sizing, and genotyping are:
•
DNA Control 007—A positive control for evaluating the eciency of the amplification step and STR
genotyping using the GlobalFiler™ Express Allelic Ladder. DNA Control 007 is present in the kit. See
“DNA Control 007 profile” on page 13.
•
GeneScan™ 600 LIZ™ Size Standard v2.0—Used for obtaining sizing results. This standard, which
has been evaluated as an internal size standard, yields precise sizing results for PCR products.
Order the GeneScan™ 600 LIZ™ Size Standard v2.0 (Cat. No. 4408399) separately.
•
GlobalFiler™ Express Allelic Ladder—Developed for accurate characterization of the alleles
amplified by the kit. The Allelic Ladder is present in the kit and allows automatic genotyping of
most of the reported alleles for the loci in the kit. See “Allelic ladder profile” on page 12.
18, 19
20, 23
GlobalFiler™ Express PCR Amplification Kit User Guide
11
Chapter 1 Product information
1
Product description
Allelic ladder profile
Figure 1 GeneMapper™ ID‑X Software plot of the GlobalFiler™ Express Allelic Ladder
12
GlobalFiler™ Express PCR Amplification Kit User Guide
DNA Control 007 profile
Chapter 1 Product information
Product description
1
Figure 2 DNA Control 007 (1 ng) amplified with the GlobalFiler™ Express PCR Amplification Kit and analyzed on
an Applied Biosystems™ 3500xL Genetic Analyzer (Y-axis scale 0 to 8,000 RFU).
GlobalFiler™ Express PCR Amplification Kit User Guide
13
Chapter 1 Product information
1
Contents and storage
Contents and storage
The GlobalFiler™ Express PCR Amplification Kit contains sucient quantities of the following reagents
to perform 200 (Cat. No. 4476609) or 1,000 (Cat. No. 4474665) amplifications at 15 μL/amplification.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set,
amplified DNA, allelic ladder, and size standard from light when not in use.
IMPORTANT! Do not refreeze kit components after thawing.
Contents Description
GlobalFiler™ Express
Master Mix
Master Mix Additive Reagent for one-time
GlobalFiler™ Express
Primer Set
Contains enzyme, salts,
dNTPs, bovine serum
albumin, enzyme, and
0.05% sodium azide in
buer and salt.
addition to the
GlobalFiler™ Express
Master Mix following first
thaw.
Contains forward and
reverse primers to
amplify human DNA
targets.
200 reactions
(Cat. No.
4476609)
1 × 1.13 mL 1 × 5.64 mL −25°C to −15°C on receipt.
1 × 0.1 mL 1 × 0.45 mL −25°C to −15°C on receipt.
1 × 1.2 mL 1 × 6 mL −25°C to −15°C on receipt.
1,000 reactions
(Cat. No.
4474665)
Storage
2°C to 8°C after first use,
for up to 6 months or up
to the expiration date stated
on the kit (whichever comes
first).
Discard the tube after
adding to the master mix.
2°C to 8°C after first use,
for up to 6 months or up
to the expiration date stated
on the kit (whichever comes
first).
GlobalFiler™ Express
Allelic Ladder
14
Contains amplified
alleles.
See “Allelic ladder
profile” on page 12 for
information.
Store protected from light.
1 × 0.065 mL 1 × 0.15 mL −25°C to −15°C on receipt.
2°C to 8°C after first use, up
to the expiration date stated
on the kit.
Store protected from light.
GlobalFiler™ Express PCR Amplification Kit User Guide
(continued)
Chapter 1 Product information
Required materials not supplied
1
200 reactions
Contents Description
DNA Control 007 Contains 2 ng/µL human
male genomic DNA from
cell line in 0.05% sodium
azide and buer
See “DNA Control 007
profile” on page 13 for
information.
[1]
DNA Control 007 is included at a concentration that is appropriate for use as an amplification control (that is, to provide confirmation of the
capability of the kit reagents to generate a profile of expected genotype). It is not designed for use as a DNA quantification control. If you
quantify aliquots of Control 007, the concentration may differ from the labeled concentration.
[1]
(Cat. No.
4476609)
1 × 0.05 mL 1 × 0.1 mL −25°C to −15°C on receipt.
1,000 reactions
(Cat. No.
4474665)
Storage
2°C to 8°C after first use, up
to the expiration date stated
on the kit.
Required materials not supplied
See Appendix B, “Materials required but not supplied”.
Instrument and software compatibility
Instrument
type
Thermal
cyclers
Validated models
•
ProFlex™ 96‑well PCR System (Cat. No. 4484075)
•
ProFlex™ 2 × 96‑well PCR System (Cat. No. 4484076)
•
Veriti™ 96‑Well Thermal Cycler (Cat. No. 4479071)
•
GeneAmp™ PCR System 9700, 96-Well Silver (Cat. No. N8050001)
•
GeneAmp™ PCR System 9700, 96-Well Gold-Plated (Cat. No. 4314878)
IMPORTANT! GlobalFiler
Veriti™ Fast 96‑Well Thermal Cycler (Cat. No. 4375305)
·
GeneAmp™ PCR System 9700, 96-Well Aluminum (Cat. No. 4314879)
·
™
Express PCR Amplification Kit is NOT validated for use with:
GlobalFiler™ Express PCR Amplification Kit User Guide
15
Chapter 1 Product information
1
Instrument and software compatibility
(continued)
Instrument
type
Genetic
analyzers
Validated models
3500/3500xL Genetic Analyzer
[1]
•
3500 Series Data Collection Software 1 (Windows™ Vista operating system) and HID Updater
3500 Data Collection Software v2 (Cat. No. 4480670)
•
3500 Series Data Collection Software 2 (Windows™ 7 operating system) and HID Updater
3500 Data Collection Software v2 (Cat. No. 4480670)
•
3500 Series Data Collection Software 3 (Windows™ 7 operating system)
•
3500 Series Data Collection Software 3.1 (Windows™ 7 operating system)
•
3500 Series Data Collection Software 4 (Windows™ 10 operating system)
•
3500 Series HID Data Collection Software v4.0.1 (Windows™ 10 operating system)
3130/3130xl Genetic Analyzer
•
3130 Series Data Collection Software 4 (Windows™ 7 operating system)
•
3130/3730 Data Collection 4 6-Dye Module v1
3730/3730xl DNA Analyzer
•
3730 Series Data Collection Software 4 (Windows™ 7 operating system)
•
3730 Series Data Collection Software 4 6-Dye Module v1
•
3730xl Data Collection Software 5 (Windows™ 10 operating system)
Note: For information on using the 3730xl DNA Analyzer, see the 3730xl Data
Collection Software 5 for HID User Bulletin: New Features and Developmental Validation
(Pub. No. MAN0019461)
SeqStudio™ Genetic Analyzer
•
SeqStudio™ Data Collection Software v1.2
•
SeqStudio™ Data Collection Software v1.2.1
Analysis
software
[1]
We conducted validation studies using the 3130xl, 3500, 3500xL, and 3730xl instruments. For validation information on the 3730xl instrument,
see the 3730xl Data Collection Software 5 for HID User Bulletin: New Features and Developmental Validation (Pub. No. MAN0019461).
GeneMapper™ ID‑X Software v1.4 or later
Windows™ XP, Windows™ 7, or Windows™ 10 operating system
16
GlobalFiler™ Express PCR Amplification Kit User Guide
Workflow
Chapter 1 Product information
Workflow
1
Perform PCR on treated or untreated paper
substrates
“Prepare the amplification kit reactions: treated paper
substrate” on page 21 or
“Prepare the amplification kit reactions: untreated
paper substrate” on page 23
▼ ▼
Obtain punch with Harris Manual Punch or BSD Semi-
Automated Dried Sample Punch Instrument
▼
Untreated paper only: Process with Prep‑n‑Go™ Buer Lyse in Prep‑n‑Go™ Buer
▼ ▼
Process with the GlobalFiler™ Express PCR
Amplification Kit
▼ ▼
Amplify with a recommended thermal cycler
“Prepare the reactions: swab substrate” on page 26
Perform PCR on swab substrates
Process with the GlobalFiler™ Express PCR
Amplification Kit
▼
Perform electrophoresis
“Set up the SeqStudio™ instruments for electrophoresis (before first use of the kit)” on page 30 or
“Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit)” on page 32 or
“Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit)” on page 36 or
“Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit)” on page 38
▼
“Prepare samples for electrophoresis” on page 41
▼
Analyze data
“Set up the GeneMapper™ ID‑X Software for analysis (before first use of the kit)” on page 44
“Create an analysis method” on page 49
“Create a size standard definition file if needed” on page 57
“Analyze and edit sample files with GeneMapper™ ID‑X Software” on page 60
“Examine or edit a project” on page 61
GlobalFiler™ Express PCR Amplification Kit User Guide
17
Perform PCR
2
Optimize PCR cycle number (before first use of the kit) .................................... 18
■
Before you begin ..................................................................... 19
■
Treated paper substrates: prepare the amplification kit reactions ........................... 20
■
Untreated paper substrates: prepare the amplification kit reactions ......................... 22
■
Swab substrates: prepare the amplification kit reactions ................................... 24
■
Perform PCR ......................................................................... 28
■
Optimize PCR cycle number (before first use of the kit)
Before using the GlobalFiler™ Express PCR Amplification Kit for the first time, perform a single initial
sensitivity experiment to determine the appropriate cycle number to use during internal validation
studies and operational use of the kit. This experiment accounts for instrument‑to‑instrument and
sample‑to‑sample variations. If you are processing multiple sample type and substrate combinations
(for example, buccal samples on treated paper and buccal samples on swabs), perform separate
sensitivity experiments for each sample type and substrate to be used for testing.
Procedural guidelines when optimizing PCR cycle number
•
(Recommended) Use 26 samples so that you can complete electrophoresis using a single 96‑well
plate. This minimizes the impact of run‑to‑run variation on the results. Examples of PCR and
electrophoresis plate layouts are provided on page 145.
•
To maximize result quality, prepare and amplify Plate 1, then repeat for Plates 2 and 3. Do not
prepare all 3 plates before amplification.
•
To minimize the eect of instrument‑to‑instrument variation, use the same thermal cycler to amplify
all 3 plates.
Select samples and prepare plates
1.
Select 26 of each sample+substrate type. Ensure that the selected samples represent a "typical"
range of samples analyzed in your laboratory.
2.
Prepare the samples and the reactions as described in the appropriate protocols later in this
chapter. Prepare sucient PCR reagents to complete amplification of three replicate plates.
3.
Create the first of 3 identical PCR plates (see page 145 for a suggested plate layout).
18
GlobalFiler™ Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Before you begin
4.
Amplify each plate using a dierent cycle number to determine the optimum conditions for use in
your laboratory.
Suggested cycle numbers for dierent sample type and substrate combinations are listed in the
following table.
2
Sample type
Blood 25, 26, 27 cycles 25, 26, 27 cycles N/A
Buccal 26, 27, 28 cycles 26, 27, 28 cycles 25, 26, 27 cycles
Treated paper Untreated paper Swab
Determine optimum PCR conditions
1.
Run the PCR products on the appropriate CE platform using the recommended protocol that is
described in Chapter 3, “Perform electrophoresis”.
2.
Based on the results of the sensitivity study, select the appropriate PCR cycle number for future
experiments.
Our studies indicate the optimum PCR cycle number should generate profiles with the following
heterozygote peak heights, with no instances of allelic dropout and minimal occurrence of o‑scale
allele peaks:
Instrument
3500 Series 3,000–12,000 RFU
3130 Series 1,000–3,000 RFU
Substrate
Heterozygous peak height
3730 Series 3,000–12,000 RFU
SeqStudio™ Genetic Analyzer 3,000–12,000 RFU
When amplifying single‑source, unpurified samples, you will see greater sample‑to‑sample variation in
peak height than you see with purified samples. Careful optimization of the cycle number helps to
minimize this variation.
Before you begin
Thaw reagents and prepare Master Mix (before first use of the kit)
1.
Thaw the Master Mix, Master Mix Additive, and Primer Set, then vortex for 3 seconds.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer
set, amplified DNA, allelic ladder, and size standard from light when not in use.
IMPORTANT! Thawing is required only during first use of the kit. After first use, reagents are
stored at 2 to 8°C and, therefore, do not require subsequent thawing. Do not refreeze the reagents.
GlobalFiler™ Express PCR Amplification Kit User Guide
19
Chapter 2
2
Treated paper substrates: prepare the amplification kit reactions
2.
3.
4.
5.
6.
Perform PCR
Before opening the tubes or bottles, remove droplets from the caps by centrifuging the tubes
briefly and tapping the bottles on the bench.
Add the following volumes of Master Mix Additive to the Master Mix:
Kit Master Mix Additive volume
200 reactions 80 µL
1,000 reactions 390 µL
Gently invert the Master Mix tube 10 times, then centrifuge the tube briefly or tap the bottle on the
bench.
Mark the cap of the Master Mix with a (+) to indicate that the Master Mix Additive has been added.
Discard the Master Mix Additive tube.
Treated paper substrates: prepare the amplification kit
reactions
Sample preparation guidelines: treated paper substrate
•
Do not add water to the wells on the reaction plate before adding the punches. If you observe
static issues with the paper discs, you can prepare and dispense the 15-µL reaction mix into the
wells of the reaction plate before adding the punches.
Alternatively, dispense 3 µL of low-TE Buer into each sample and negative amplification control
well (NOT the positive amplification control wells) before adding the punches.
•
Make the punch as close as possible to the center of the sample to ensure optimum peak intensity.
Increasing the size of the punch may cause inhibition during PCR amplification.
•
For manual punching: Place the tip of a 1.2 mm Harris Micro-Punch on the card, hold the barrel of
the Harris Micro-Punch (do not touch the plunger), gently press and twist 1/4-turn, then eject the
punch in to the appropriate well on the reaction plate.
•
For automated punching: See the User Guide of your automated or semiautomated disc punch
instrument for proper guidance.
Prepare low-TE buer
For optimal results, we recommend using low-TE buer for sample preparation. Prepare it as described
in this procedure or buy it from Teknova (Cat. No. T0223).
20
1.
Mix together:
•
•
•
10 mL of 1 M Tris-HCl, pH 8.0
0.2 mL of 0.5 M EDTA, pH 8.0
990 mL glass-distilled or deionized water
Note: Adjust the volumes accordingly for specific needs.
GlobalFiler™ Express PCR Amplification Kit User Guide
Chapter 2
Treated paper substrates: prepare the amplification kit reactions
2.
Aliquot, then autoclave the solutions.
3.
Store the aliquots at room temperature.
Prepare the amplification kit reactions: treated paper substrate
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set,
amplified DNA, allelic ladder, and size standard from light when not in use.
If this is the first time you are using the kit, follow the instructions in “Thaw reagents and prepare Master
Mix (before first use of the kit)” on page 19 before proceeding.
1.
Add samples to the MicroAmp™ Optical 96‑well Reaction Plate:
Perform PCR
2
To these wells of the plate ...
Negative control 1.2 mm blank disc
Test samples 1.2 mm sample disc
Positive control
IMPORTANT! Do not add a blank disc
to the positive control well.
Note: The volumes of positive control are suggested amounts and can be adjusted if peak heights
are too high or too low for your optimized cycle number.
2.
Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove
droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
3.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix 6.0 μL
Primer Set 6.0 μL
Low-TE buer 3.0 μL
For 25 and 26 cycles 3 μL of Control DNA 007
For 27 cycles 2 μL of Control DNA 007
For 28 cycles 1 μL of Control DNA 007
Add...
Volume per reaction
Note: Include volume for additional reactions to provide excess volume for the loss that occurs
during reagent transfers.
IMPORTANT! This kit is optimized for a 15-μL PCR volume to overcome the PCR inhibition that is
expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the
ability of the kit chemistry to generate full STR profiles.
4.
Vortex the reaction mix for 3 seconds, then centrifuge briefly.
5.
Dispense 15 µL of the reaction mix into each reaction well of a MicroAmp™ Optical 96-Well
Reaction Plate.
GlobalFiler™ Express PCR Amplification Kit User Guide
21
1
Chapter 2 Perform PCR
2
Untreated paper substrates: prepare the amplification kit reactions
6.
Seal the plate with MicroAmp™ Clear Adhesive Film (Cat. No. 4306311) or MicroAmp™ Optical
Adhesive Film (Cat. No. 4311971).
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across
all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across
all wells.
IMPORTANT! If you are using the GeneAmp
block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp™ Optical
Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during
thermal cycling. Other validated thermal cyclers do not require a compression pad.
7.
Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
8.
Amplify the samples as described in Chapter 2, “Perform PCR”.
IMPORTANT! This kit is not validated for use with the GeneAmp
aluminum 96-well block. Use of this thermal cycling platform may adversely aect performance of
this kit.
™
PCR System 9700 with silver or gold-plated silver
™
PCR System 9700 with the
Untreated paper substrates: prepare the amplification kit
reactions
Sample preparation guidelines: untreated paper substrate
•
Make a 1.2 mm punch as close as possible to the center of the sample to ensure optimum peak
intensity. Increasing the size of the punch may cause inhibition during PCR amplification.
•
If you are using a Bode Buccal DNA Collector™, make
a 1.2 mm punch as close as possible to the tip of
the DNA collector to ensure optimum peak intensity.
A larger punch may cause inhibition during PCR
amplification.
•
For manual punching: Place the tip of a 1.2 mm Harris
Micro-Punch on the card, hold the barrel of the Harris
Micro-Punch (do not touch the plunger), gently press
and twist 1/4-turn, then eject the punch in to the
appropriate well on the reaction plate.
•
For automated punching: See the User Guide of your
automated or semiautomated disc punch instrument for
proper guidance.
1
Location of punch with a Bode Buccal
DNA Collector
™
22
GlobalFiler™ Express PCR Amplification Kit User Guide
Chapter 2 Perform PCR
Untreated paper substrates: prepare the amplification kit reactions
Prepare the amplification kit reactions: untreated paper substrate
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set,
amplified DNA, allelic ladder, and size standard from light when not in use.
If this is the first time you are using the kit, follow the instructions in “Thaw reagents and prepare Master
Mix (before first use of the kit)” on page 19 before proceeding.
1.
Add Prep‑n‑Go™ Buer (Cat. No. 4467079) to the MicroAmp™ Optical 96-Well Reaction Plate:
To these wells ... Add...
Negative control 3 μL of Prep‑n‑Go™ Buer
Test samples 3 μL of Prep‑n‑Go™ Buer
Positive control For 25 and 26 cycles 0 μL of Prep‑n‑Go™ Buer
For 27 cycles 1 μL of Prep‑n‑Go™ Buer
For 28 cycles 2 μL of Prep‑n‑Go™ Buer
2
2.
Add samples to the reaction plate:
To these wells ...
Negative control 1.2 mm blank disc
Test samples 1.2 mm sample disc
Positive control
IMPORTANT! Do not add a blank disc
to the positive control well.
Note: The volumes of positive control are suggested amounts and may be adjusted if peak
heights are too high or too low for your optimized cycle number.
3.
Centrifuge the plate to ensure that the punches are immersed in the Prep‑n‑Go™ Buer.
4.
Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove
droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
5.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix 6.0 μL
For 25 and 26 cycles 3 μL of Control DNA 007
For 27 cycles 2 μL of Control DNA 007
For 28 cycles 1 μL of Control DNA 007
Add...
Volume per reaction
Primer Set 6.0 μL
GlobalFiler™ Express PCR Amplification Kit User Guide
23
Chapter 2
2
Swab substrates: prepare the amplification kit reactions
Perform PCR
Note: Include volume for additional reactions to provide excess volume for the loss that occurs
during reagent transfers.
IMPORTANT! This kit is optimized for a 15-μL PCR volume to overcome the PCR inhibition that is
expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the
ability of the kit chemistry to generate full STR profiles.
6.
Vortex the reaction mix for 3 seconds, then centrifuge briefly.
7.
Dispense 12 µL of the reaction mix into each reaction well of a MicroAmp™ Optical 96-Well
Reaction Plate.
The final volume in each well is 15 µL (reaction mix plus Prep‑n‑Go™ Buer and sample or positive
control).
8.
Seal the plate with MicroAmp™ Clear Adhesive Film (Cat. No. 4306311) or MicroAmp™ Optical
Adhesive Film (Cat. No. 4311971).
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across
all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across
all wells.
IMPORTANT! If you are using the GeneAmp
block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp™ Optical
Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during
thermal cycling. Other validated thermal cyclers do not require a compression pad.
9.
Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
10.
Amplify the samples as described in Chapter 2, “Perform PCR”.
IMPORTANT! This kit is not validated for use with the GeneAmp
aluminum 96-well block. Use of this thermal cycling platform may adversely aect performance of
this kit.
™
PCR System 9700 with silver or gold-plated silver
™
PCR System 9700 with the
Swab substrates: prepare the amplification kit reactions
Sample preparation guidelines: swab substrate
•
Detach each buccal swab head from the swab shaft before lysis.
•
If you are using the heated lysis protocol, perform lysis in either of the following formats:
–
1.5-mL tubes with a heat block (VWR™ Scientific Select dry heat block or similar)
–
PrepFiler™ 96-Well Processing Plates (Cat. No. A47010)
–
Robbins Scientific™ Model 400 Hybridization Incubator or similar
24
GlobalFiler™ Express PCR Amplification Kit User Guide
Swab substrates: prepare the amplification kit reactions
–
Agilent™ Benchtop Rack for 200 μL Tubes/V Bottom Plates (metal) or similar (Cat. No. 410094)
IMPORTANT! Do not use a plastic plate adaptor.
•
For optimum performance, lyse the entire swab. If you need to preserve the sample, use half of the
lysate prepared from the entire swab.
Prepare the sample lysate: room temperature
This protocol may improve the performance for challenging or aged samples.
1.
Add 400 µL Prep‑n‑Go™ Buer (Cat. No. 4471406) to 1.5-mL tubes or the appropriate wells of a
PrepFiler™ 96-Well Processing Plate (Cat. No. A47010).
2.
Into each tube or well, put the entire head of each swab, then let stand for 20 minutes at room
temperature (20°C to 25°C) to lyse the sample.
3.
After 20 minutes, transfer the sample lysate out of the sample plate into tubes or plates for storage,
then discard the deep‑well plate containing the swab heads.
Note: To minimize the risk of contamination, do not remove the swab heads from the sample
lysate plate before transferring the lysate.
Chapter 2
Perform PCR
2
4.
Go to “Prepare the reactions: swab substrate” on page 26 or “Store the sample lysate” on
page 27.
Prepare the sample lysate: heat protocol
This protocol may improve the performance for challenging or aged samples.
1.
Preheat the heat block to 90°C or the oven with metal plate adaptor to 99°C.
2.
Add 400 µL Prep‑n‑Go™ Buer (for buccal swabs, Cat. No. 4471406) to 1.5-mL tubes or the
appropriate wells of a PrepFiler™ 96-Well Processing Plate (Cat. No. A47010).
3.
Into each tube or well, put the entire head of each swab. If you are using tubes, cap the tubes. Let
the tubes or plate stand for 20 minutes in the preheated heat block or oven to lyse the sample.
4.
After 20 minutes, remove the tubes or the deep‑well plate from the heat block or oven.
5.
Let the lysate stand at room temperature for at least 15 minutes to cool the lysate (for accurate
pipetting).
6.
Transfer the sample lysate out of the 1.5-mL tubes or sample plate into tubes or plates for storage.
Discard the 1.5-mL tubes or deep‑well plate containing the swab heads.
Note: To minimize the risk of contamination, do not remove the swab heads from the sample
lysate plate before transferring the lysate.
7.
Go to “Prepare the reactions: swab substrate” on page 26 or “Store the sample lysate” on
page 27.
GlobalFiler™ Express PCR Amplification Kit User Guide
25
Chapter 2 Perform PCR
2
Swab substrates: prepare the amplification kit reactions
Prepare the reactions: swab substrate
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set,
amplified DNA, allelic ladder, and size standard from light when not in use.
If this is the first time you are using the kit, follow the instructions in “Thaw reagents and prepare Master
Mix (before first use of the kit)” on page 19 before proceeding.
1.
Add Prep‑n‑Go™ Buer (Cat. No. 4471406) to the control wells in the MicroAmp™ Optical 96-Well
Reaction Plate:
To these wells ... Add...
Negative control 3 μL of Prep‑n‑Go™ Buer
Positive control For 25 and 26 cycles 0 μL of Prep‑n‑Go™ Buer
2.
Vortex the Master Mix and Primer Set for 3 seconds. Before opening the tubes or bottles, remove
droplets from the caps by centrifuging the tubes briefly or tapping the bottles on the bench.
For 27 cycles 1 μL of Prep‑n‑Go™ Buer
For 28 cycles 2 μL of Prep‑n‑Go™ Buer
3.
Pipet the required volumes of components into an appropriately sized polypropylene tube.
Reaction component
Master Mix 6.0 μL
Primer Set 6.0 μL
Note: Include volume for additional reactions to provide excess volume for the loss that occurs
during reagent transfers.
Volume per reaction
IMPORTANT! This kit is optimized for a 15-μL PCR volume to overcome the PCR inhibition that is
expected when amplifying unpurified samples. Using a lower PCR reaction volume may reduce the
ability of the kit chemistry to generate full STR profiles.
4.
Vortex the reaction mix for 3 seconds, then centrifuge briefly.
5.
Dispense 12 μL of the reaction mix into each reaction well of a MicroAmp™ Optical 96-Well
Reaction Plate.
The final volume in each well is 15 μL (reaction mix plus Prep‑n‑Go™ Buer or sample lysate or
positive control).
26
GlobalFiler™ Express PCR Amplification Kit User Guide
6.
Add samples to the reaction plate:
Chapter 2 Perform PCR
Swab substrates: prepare the amplification kit reactions
2
To these well(s) of a MicroAmp™ Optical 96-
Well Reaction Plate...
Test samples 3 μL of sample lysate
Positive control For 25 and 26 cycles 3 μL of Control DNA
For 27 cycles 2 μL of Control DNA
For 28 cycles 1 μL of Control DNA
Add...
007
007
007
Note: The volumes of positive control are suggested amounts and may be adjusted if peak
heights are too high or too low for your optimized cycle number.
7.
Seal the plate with MicroAmp™ Clear Adhesive Film (Cat. No. 4306311) or MicroAmp™ Optical
Adhesive Film (Cat. No. 4311971).
IMPORTANT! We recommend adhesive film for plate sealing to provide a consistent seal across
all wells and prevent evaporation. Do not use caps, which may not provide a consistent seal across
all wells.
IMPORTANT! If you are using the GeneAmp
™
PCR System 9700 with silver or gold-plated silver
block and adhesive clear film instead of caps to seal the plate wells, place a MicroAmp™ Optical
Film Compression Pad (Cat. No. 4312639) on top of the plate to prevent evaporation during
thermal cycling. Other validated thermal cyclers do not require a compression pad.
8.
Vortex the reaction mix at medium speed for 3 seconds.
9.
Centrifuge the plate at 3,000 rpm for about 20 seconds in a tabletop centrifuge with plate holders.
10.
Amplify the samples as described in Chapter 2, “Perform PCR”.
IMPORTANT! This kit is not validated for use with the GeneAmp
aluminum 96-well block. Use of this thermal cycling platform may adversely aect performance of
this kit.
Store the sample lysate
1.
Cap the sample lysate storage tubes or seal the sample lysate storage plate with MicroAmp™ Clear
Adhesive Film.
2.
Store the sample lysate as needed:
If you are storing the sample lysate...
™
PCR System 9700 with the
Then place at...
<2 weeks 2°C to 8°C
>2 weeks –25°C to –15°C
GlobalFiler™ Express PCR Amplification Kit User Guide
27
Chapter 2 Perform PCR
2
Perform PCR
Note: The eects of multiple freeze/thaw cycles on the lysate have not been fully evaluated.
Therefore, multiple freeze/thaw cycles are not recommended.
Perform PCR
IMPORTANT! This kit is validated for use with the thermal cyclers listed in “Instrument and software
compatibility” on page 15.
1.
Program the thermal cycling conditions.
IMPORTANT! If you are using the GeneAmp
If you are using the ProFlex™ 96‑well PCR System, select the GeneAmp™ PCR System 9700
simulation mode. If you are using the Veriti™ Thermal Cycler, select the 100% ramping rate. Do not
use 9600 emulation mode.
™
PCR System 9700, select the Max ramping mode.
Initial incubation
step
HOLD CYCLE HOLD HOLD
95°C, 1 minute 94°C, 3 seconds 60°C, 30 seconds 60°C, 8 minutes 4°C, up to 24 hours
[1]
See “Optimize PCR cycle number (before first use of the kit)” on page 18.
[2]
The infinity (∞) setting allows an unlimited hold time.
2.
Load the plate into the thermal cycler, close the heated cover, then start the run.
Optimum cycle number
Denature Anneal/Extend
[1]
Final extension Final hold
IMPORTANT! If you are using adhesive clear film instead of caps to seal the plate wells, be
sure to place a MicroAmp™ Optical Film Compression Pad (Cat. No. 4312639) on top of the plate
to prevent evaporation during thermal cycling. The Veriti™ Thermal Cycler, ProFlex™ 96‑well PCR
System, and ProFlex™ 2 × 96‑well PCR System do not require a compression pad.
3.
When the run is complete, store the amplified DNA.
If you are storing the DNA...
<2 weeks 2°C to 8°C
>2 weeks –25°C to –15°C
Then place at...
IMPORTANT! Protect the amplified DNA from light.
[2]
28
GlobalFiler™ Express PCR Amplification Kit User Guide
Perform electrophoresis
3
Allelic ladder requirements for electrophoresis ........................................... 29
■
Materials required for electrophoresis ................................................... 30
■
Set up the SeqStudio™ instruments for electrophoresis (before first use of the kit) ............ 30
■
Set up the 3500/3500xL instruments for electrophoresis (before first use of the kit) ........... 32
■
Set up the 3130/3130xl instruments for electrophoresis (before first use of the kit) ............ 36
■
Set up the 3730/3730xl instruments for electrophoresis (before first use of the kit) ............ 38
■
Prepare samples for electrophoresis .................................................... 41
■
Allelic ladder requirements for electrophoresis
To accurately genotype samples, you must run an allelic ladder with the samples.
Instrument
3500 1 per 3 injections 8 samples 23 samples + 1 allelic ladder
3500xl 1 per injection 24 samples 23 samples + 1 allelic ladder
3130 1 per 4 injections 4 samples 15 samples + 1 allelic ladder
3130xl 1 per injection 16 samples 15 samples + 1 allelic ladder
3730/3730xl,
48‑capillary
SeqStudio
IMPORTANT! Variation in laboratory temperature can cause changes in fragment migration speed and
sizing variation between runs. Follow the guidelines in the preceding table, which should account for
normal variation in run speed. Perform internal validation studies to verify the required allelic ladder
injection frequency, to ensure accurate genotyping of all samples in your laboratory environment.
It is critical to genotype using an allelic ladder run under the same conditions as the samples. Size
values obtained for the same sample can dier between instrument platforms, because of dierent
polymer matrices and electrophoretic conditions.
™
Number of allelic
ladders to run
3 per injection 48 samples 15 samples + 1 allelic ladder
1 per 6 injections 4 samples 23 samples + 1 allelic ladder
One injection
equals
Number of samples per allelic ladder(s)
GlobalFiler™ Express PCR Amplification Kit User Guide
29
Chapter 3 Perform electrophoresis
3
Materials required for electrophoresis
Materials required for electrophoresis
Appendix B, “Materials required but not supplied” lists the required materials that are not supplied with
this kit.
IMPORTANT! The fluorescent dyes attached to the primers are light-sensitive. Protect the primer set,
amplified DNA, allelic ladder, and size standard from light when not in use.
Set up the SeqStudio™ instruments for electrophoresis
(before first use of the kit)
Electrophoresis software setup
The following table lists the data collection software and the run modules that you can use to analyze
PCR products generated by this kit. For details on the procedures, see the documents listed in
“Documentation and support” on page 151.
Genetic
Analyzer
SeqStudio
Data Collection
Software
™
SeqStudio
Data Collection
Software v1.2.1
Additional software Run modules and conditions
™
None
•
Run Module: HIDAnalysis
•
Injection Conditions: 1.2 kV/10 sec
•
Run Conditions: 11 kV/1,120 sec
•
Dye Set J6
•
Kit: GlobalFiler Express (to enable marker-to-marker
pull-up reduction feature)
30
GlobalFiler™ Express PCR Amplification Kit User Guide
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