iPLEX Gold Application Guide-111006 - University of Chicagocancer-seqbase.uchicago.edu/documents/iPLEXGold... · 2006. 11. 13. · Title: iPLEX Gold Application Guide-111006.book

i P L E X ™Gold

A p p l i c a t i o n G u i d e

Contents

November 10, 2006 iiPLEX™ Gold Application GuideDoc. 11555 R00, CO 060190 (Matrix, Samsung, Compact)

Chapter 4Processing

Low Plex iPLEXGold Reactions

for OneDestination

Plate (1plex -18plex)

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Daily and Weekly Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Daily Tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Weekly Tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Preparing and Adding SAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19To prepare the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . . 19To add the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21To incubate the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Adjusting, Preparing, and Adding the Low Plex iPLEX Gold Reaction Cocktail 24To adjust the extension primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24To prepare the Low Plex iPLEX Gold reaction cocktail (same multiplexed as-

says, different DNAs for 96 to 384 dispensing) . . . . . . . . . . . . . . . . . . . 24To prepare the Low Plex iPLEX Gold reaction cocktail (same DNA, different

multiplexed assays for 96 to 384 dispensing) . . . . . . . . . . . . . . . . . . . . 26To add the low plex iPLEX Gold reaction cocktail (96 to 384 dispensing) 27

Thermocycling the Low Plex iPLEX Gold Reaction . . . . . . . . . . . . . . . . . . . . . 29To thermocycle the low plex iPLEX Gold reaction . . . . . . . . . . . . . . . . . . 29

Cleaning Up the Low Plex iPLEX Gold Reaction Products . . . . . . . . . . . . . . . . 31To prepare a plate of resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31To add water to the 384-well sample microtiter plate . . . . . . . . . . . . . . . 32To add resin to the sample microtiter plate . . . . . . . . . . . . . . . . . . . . . . . 33To rotate and centrifuge the low plex iPLEX Gold reaction products . . . 34

Nanodispensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Defining Assays and Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Acquiring Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Chapter 5Processing

High PlexiPLEX Gold

Reactions forOne

DestinationPlate (19plex -

36+plex)

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37


Preparing and Adding SAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39To prepare the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . . 39To add the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41To incubate the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Adjusting, Preparing, and Adding the High Plex iPLEX Gold Reaction Cocktail 44

iPLEX™ Gold Application Guide ii November 10, 2006(Matrix, Samsung, Compact) Doc. 11548 R03, CO 060150

To adjust the extension primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44To prepare the High Plex iPLEX Gold reaction cocktail (same multiplexed

assays, different DNAs for 96 to 384 dispensing) . . . . . . . . . . . . . . . . . 44To prepare the High Plex iPLEX Gold reaction cocktail (same DNA, different

multiplexed assays for 96 to 384 dispensing) . . . . . . . . . . . . . . . . . . . . 46To add the High Plex iPLEX Gold reaction cocktail (96 to 384 dispensing)

47

Thermocycling the High Plex iPLEX Gold Reaction . . . . . . . . . . . . . . . . . . . . . 49To thermocycle the High Plex iPLEX Gold reaction . . . . . . . . . . . . . . . . 49

Cleaning Up the High Plex iPLEX Gold Reaction Products . . . . . . . . . . . . . . . . 51To prepare a plate of resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51To add water to the 384-well sample microtiter plate . . . . . . . . . . . . . . . 52To add resin to the sample microtiter plate . . . . . . . . . . . . . . . . . . . . . . . 53To rotate and centrifuge the high plex iPLEX Gold reaction products . . . 54

Nanodispensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55


Acquiring Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Chapter 6ProcessingLow iPLEX

Gold Reactionsfor Two

DestinationPlates(1plex -

18plex)

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57


Preparing and Adding SAP to Two Destination Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

To prepare the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . . 59To add the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61To incubate the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Adjusting, Preparing, and Adding the Low Plex iPLEX Gold Reaction Cocktail 63To adjust the extension primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63To prepare the low plex iPLEX Gold reaction cocktail (same multiplexed as-

says, different DNAs for 96 to 384 dispensing) . . . . . . . . . . . . . . . . . . 63To prepare the low plex iPLEX Gold reaction cocktail (same DNA, different

multiplexed assays for 96 to 384 dispensing) . . . . . . . . . . . . . . . . . . . . 65To add the low plex iPLEX Gold reaction cocktail (96 to 384 dispensing) 66

Thermocycling the low plex iPLEX Gold Reaction in Two Plates . . . . . . . . . . . 68To thermocycle the low plex iPLEX Gold reaction . . . . . . . . . . . . . . . . . 68

November 10, 2006 iiiiPLEX™ Gold Application GuideDoc. 11555 R00, CO 060190 (Matrix, Samsung, Compact)

Cleaning Up the Low Plex iPLEX Gold Reaction Products . . . . . . . . . . . . . . . . 70To prepare a plate of resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70To add water to the 384-well sample microtiter plate . . . . . . . . . . . . . . . 71To add resin to the sample microtiter plate . . . . . . . . . . . . . . . . . . . . . . . 73To rotate and centrifuge the low plex iPLEX Gold reaction products . . . 74

Nanodispensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74



Chapter 7ProcessingHigh iPLEX

Gold Reactionsfor Two

DestinationPlates(19plex -

36+plex)

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75


Preparing and Adding SAP to Two Destination Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

To prepare the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . . 77To add the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79To incubate the SAP enzyme solution: . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Adjusting, Preparing, and Adding the High Plex iPLEX Gold Reaction Cocktail 81To adjust the extension primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81To prepare the high plex iPLEX Gold reaction cocktail (same multiplexed as-

says, different DNAs for 96 to 384 dispensing) . . . . . . . . . . . . . . . . . . . 81To prepare the high plex iPLEX Gold reaction cocktail (same DNA, different

multiplexed assays for 96 to 384 dispensing) . . . . . . . . . . . . . . . . . . . . 83To add the high plex iPLEX Gold reaction cocktail (96 to 384 dispensing) .

84

Thermocycling the High Plex iPLEX Gold Reaction in Two Plates . . . . . . . . . . 86To thermocycle the high plex iPLEX Gold reaction . . . . . . . . . . . . . . . . . 86

Cleaning Up the High Plex iPLEX Gold Reaction Products . . . . . . . . . . . . . . . . 88To prepare a plate of resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88To add water to the 384-well sample microtiter plate . . . . . . . . . . . . . . . 89To add resin to the sample microtiter plate . . . . . . . . . . . . . . . . . . . . . . . 91To rotate and centrifuge the high plex iPLEX Gold reaction products . . . 92

Nanodispensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92



iPLEX™ Gold Application Guide iv November 10, 2006(Matrix, Samsung, Compact) Doc. 11548 R03, CO 060150

Appendix AExtension

PrimerAdjustment

Methods

Appendix BTerms andConditions

November 10, 2006 viPLEX™ Gold Application GuideDoc. 11555 R00, CO 060190 (Matrix, Samsung, Compact)

iPLEX™ Gold Application Guide vi November 10, 2006(Matrix, Samsung, Compact) Doc. 11548 R03, CO 060150

Chapter 1Introduction to Genotyping

Using ThisGuide

This manual provides information on the tasks required to run genotyping experiments. This manual assumes you are using the Matrix Liquid Handler, Samsung Nanodispenser, and the Compact Analyzer. For more information, see the following:

• MassARRAY Liquid Handler (Matrix): See subsequent chapters in this guide for instructions. See the MassARRAY Liquid Handler User’s Guide (Part Number 11541) for detailsFor users of the Beckman Multimek liquid handler, see MassARRAY Liquid Handler User’s Guide (Part Number 11527).

• MassARRAY Samsung Nanodispenser: See the MassARRAY Nanodispenser User’s Guide (Samsung) (Part Number 11537).For users of the RoboDesign Nanodispenser, see the MassARRAY Nanodispenser User’s Guide (RoboDesign) (Part Number 11536).

• MassARRAY Analyzer Compact: See the MassARRAY Analyzer Compact User’s Guide (Part Number 11533) for instructions.For users of the Bruker Autoflex, see the MassARRAY Typer User’s Guide (Autoflex) (Part Number 11530) .

For users of the Bruker Biflex, see the MassARRAY Typer User’s Guide (Biflex) (Part Number 11531).

This chapter presents an overview of the iPLEX Gold reaction, tips for designing genotyping assays, and a list of components, reagents, and consumables used for genotyping. Subsequent chapters provide instructions for processing genotyping plates on the MassARRAY Liquid Handler (Matrix).

Automated genotyping involves the following tasks:

• Isolation, quantitation, and amplification of DNA.• Preparation of iPLEX Gold reaction products using the liquid handler.• Transfer of iPLEX Gold reaction products to SpectroCHIPs using the

nanodispenser.• Defining the setup of assays and plates in the MassARRAY database using Assay

Editor and Plate Editor software.• Acquisition of spectra using the analyzer compact mass spectrometer.• Analysis of spectral data using TyperAnalyzer software.

November 10, 2006 1 iPLEX™ Gold Application GuideDoc. 11555 R00, CO 060190 (Matrix, Samsung, Compact)

Introduction to GenotypingiPLEX Gold Reaction

iPLEX GoldReaction

The iPLEX Gold reaction is a universal method for detecting insertions, deletions, substitutions, and other polymorphisms in amplified DNA. See Figure 1.

forward PCR primer

reverse PCR primer10-mer tag

[G/C][C/G]

PCR Product

SAP Treatment

iPLEX Reaction

Sample conditioning, dispensing, and MALDI-TOF MS

extension into SNP site

Amplification

CG

Primer

5’ 3’

24-plex spectrum

10-mer tag

3’ 5’

[G/C][C/G]

GC

Primer

Spectrum

extension into SNP site

Genomic DNA

SAP treatment to neutralizeunincorporated dNTPs

iPLEX Gold cocktail containing primer, enzyme, buffer, and mass-modified nucleotides

MALDI-TOF mass spectrometry analysis

Allele 1

Allele 2

Figure 1: iPLEX Gold Reaction

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Introduction to GenotypingiPLEX Gold Reaction

The first step in the post-PCR processing of iPLEX Gold reactions is to neutralize unincorporated dNTPs in amplification products using shrimp alkaline phosphatase (SAP). The SAP cleaves a phosphate from the unincorporated dNTPs, converting them to dNDPs and rendering them unavailable to future reaction.

Next, iPLEX Gold reaction cocktail (primer, enzyme, buffer, mass-modified nucleotides) is added to the amplification products.

The amplification products and iPLEX Gold reaction cocktail are thermocycled to process the iPLEX Gold reaction, which involves the enzymatic addition of mass-modified nucleotides into the diagnostic site.

In the reaction mixture, all four mass-modified nucleotides—A, T, C, and G—are present. During the iPLEX Gold reaction, the primer is extended by one of the nucleotides, which terminates the extension of the primer.

Using a DNA polymerase that incorporates nucleotides, the iPLEX Gold reaction produces allele-specific extension products of different masses depending on the sequence analyzed (see Table 1 and Figure 2).

Prior to mass spectrometry, the products of the iPLEX Gold reaction are desalted and transferred onto a SpectroCHIP by the MassARRAY nanodispenser. The SpectroCHIP is then analyzed by the MassARRAY analyzer compact.

Table 1: Example of extension products of a heterozygote assay

Analytes Peak description Length of product [bp]

Calculated mass [Da]

Unextended primer

Extension Primer 20 6163.0

Allele 1 Extension Primer + A 21 6434.2

Allele 2 Extension Primer + G 21 6450.2

Figure 2: Spectrum of A and G terminated extension products


Introduction to GenotypingGuidelines for Designing Genotyping Assays

Guidelines forDesigning

GenotypingAssays

To design an assay, follow these guidelines below.

1. Choose the SNP or mutation of interest.2. Design the amplification primers:

• Optimal amplicon size containing the polymorphic site should be 80 to 120 bp. Fragments longer than 400 bp should be avoided.

• To avoid confusion in the mass spectrum and improve PCR performance, make sure that the mass of the amplification primer is different from the extension primer and its extension products. It is recommended that you add a generic 10-mer tag to the 5’ end of each amplification primer (referred to as the 10-mer tag whose sequence is 5’-ACGTTGGATG-3’).

3. Design the extension primer:• Design an extension primer so the 3' end is immediately adjacent to the

polymorphic site.• Extension primers should be between 15 and 30 nucleotides long

(approximately 4500 to 9000 Da). Tm should be approximately 60° C or higher.

• The mass of the extended primer should be within the mass range of 4500 to 9000 Da (see Table 2).

4. Special considerations for multiplexing the iPLEX Gold reaction:• For best results, make sure that all extension primers and extension products’

masses differ by at least 30 Da (except for alleles of the same assay).• The preferred mass range for analytes is 4500 to 9000 Da.

Components The following MassARRAY components are used to process iPLEX Gold reactions.

• MassARRAY Liquid Handler (Matrix) (automated, 96-channel pipettor)• Liquid handler controller PC• Liquid handler plate flatteners• Supply and waste reservoirs• Clean resin plates, spoon, and scraper

Reagents andSpectroCHIPs

The iPLEX Gold Reagent Kit and Clean Resin Kit contain the reagents and SpectroCHIPs required to perform iPLEX Gold reactions.

For more information about the iPLEX Gold Reagent Kit, see the “iPLEX Gold Assay Product Description” (included with the iPLEX Gold Reagent Kit).

Table 2: Mass differences between the iPLEX Gold products for a single assay

Terminator A C G T

A 0 -24 16 55.9

C 24 0 40 79.9

G -16 -40 0 39.9

T -55.9 -79.9 -39.9 0


Introduction to GenotypingOther Essential Components

Other EssentialComponents

The following components are not provided with the MassARRAY system, but are required for processing iPLEX Gold reactions.

• Thermal cycler capable of processing 384-well microtiter plates• Rotator capable of rotating a microtiter plate along its long axis• Plate centrifuge• Tube centrifuge• Twelve-channel pipettor and reservoirs• Single-channel pipettors• Repeater (multistep pipette)

Consumables The following consumables are required for processing iPLEX Gold reactions on the liquid handler.

• iPLEX Gold Reagent Kit (SEQUENOM catalog #10136)• SpectroCHIP® Arrays and Clean Resin kit (SEQUENOM catalog #10117)• 0.5-30.0 μL 96-channel tips for the liquid handler (Matrix #5506)• Reagent reservoirs (Matrix #1064-05-8)• 96-Well, polystyrene microtiter plates (96-Well Plate Vee Bottom, Sarstedt, Inc.

#82.1583)• Disposable plate sealing film (Adhesive PCR Film #SP-0027)• Adhesive sealing foil (Marsh Biomedical Products, Inc. #AB-0626)• Tubes (1.5 and 5 mL)• Water (Deionized: HPLC grade)• Deionized water• Reagent-grade isopropanol (70%)• Water or mild detergent to wipe off deck and work area

Safety Protective laboratory gloves and safety glasses are required when handling any reagent.

DNA SampleRequirements

for iPLEX Gold

For genotyping, you must isolate, quantitate, and amplify the genomic DNA samples to be used with the MassARRAY system. See “Chapter 2 Isolating and Quantitating DNA” on page 7 and “Chapter 3 Amplifying DNA for Genotyping” on page 11 in this guide for information.


Introduction to GenotypingDNA Sample Requirements for iPLEX Gold

Notes:


Chapter 2Isolating and Quantitating DNA

Introduction Before conducting any reaction to determine a genetic variation such as a single nucleotide polymorphism (SNP) with the MassARRAY system, you must have stocks of highly pure DNA to use as templates for amplification using the polymerase chain reaction (PCR).

Depending on the application, genomic DNA or cDNA is used as a template to amplify the region surrounding the genetic variation(s) of interest. Amplicons for use with the MassARRAY system are typically 70-150 bp in length. Since the amplicons are relatively short, only genomic DNA is discussed in this chapter—in most cases, the information in this chapter is also applicable to cDNA. If you wish to use cDNA templates, it is assumed you have a cDNA generation method already optimized.

After isolation, and before PCR, the genomic DNA must be quantitated. We recommend quantitation be done twice—first, with an ultraviolet (UV) spectrophotometer for an initial estimate; then with PicoGreen® dsDNA quantitation reagent.

Finally, after isolation and quantitation, we recommend diluting a large aliquot of the DNA to a concentration of 50 ng/μL.

Isolating DNA DNA QualityTo produce large quantities of PCR amplicons for use with the MassARRAY system, it is recommended that the genomic-DNA template be highly pure. The ratio of ultraviolet (UV) spectrophotometer readings at 260 nm and 280 nm wavelengths should be between 1.7 and 2.0 (i.e., A260/A280 = 1.7-2.0). Ratios in this range indicate the presence of nucleic acid with low amounts of contaminating protein. In general, we have found genomic DNA of this quality is suitable for downstream applications, such as PCR, following isolation from samples such as whole blood, buffy coat, or cultured cells.

Isolation KitsOut of the several commercially available DNA-isolation kits we have tested, we found the PUREGENE™ Genomic DNA Purification Kit from Gentra Systems, Inc. to perform best. In our testing, we isolated genomic DNA from whole blood, with 1-10 mL of starting material. All kits isolated genomic DNA well enough to produce PCR amplicons suitable for use with the MassARRAY system. The kit from Gentra Systems had the highest average yield of genomic DNA per mL of blood processed, along with the best average A260/A280 ratio.

We recommend the use of the kit from Gentra Systems. More information about the PUREGENE Genomic DNA Purification Kit can be found at Gentra Systems’ Web site (www.gentra.com) or by calling their technical services at (800) 866-3039 in the United States.

Note: Genomic DNA may be isolated using any method you wish—it is not absolutely necessary you use the kit mentioned above. The only requirement is that whatever method you use yields highly pure DNA suitable for PCR.


Isolating and Quantitating DNAQuantitating DNA

Isolation Protocol

For information about protocols for using the PUREGENE Genomic DNA Purification Kit, see Genomic DNA Purification Kit Instructions included with the kit.

Note: DNA isolated with the PUREGENE Genomic DNA Purification Kit is stable for at least 9 years at 4º C. For long term storage, the DNA may be stored at -20º C, but, it is recommended that you avoid repeated freezing and thawing to reduce DNA damage. For more information about DNA stability, see Gentra Systems’ Web site (www.gentra.com).

Warning: Certain chemicals used in PUREGENE reagents may be hazardous: Tris(hydoxymethyl) aminomethane, ethylenediaminetetraacetic acid, sodium dodecyl sulfate, ammonium chloride, and ammonium acetate. These chemicals may be harmful if swallowed and contact with the eyes and skin should be avoided. In case of contact, wash with large amounts of water and seek medical attention. Wear protective clothing.

Material Safety Data Sheets (MSDS) are available from Gentra Systems, Inc.

QuantitatingDNA

With an Ultraviolet SpectrophotometerInitial quantitation should be conducted with a UV spectrophotometer at wavelengths of 260 nm and 280 nm. The ratio of absorbance readings at the two wavelengths should be between 1.7 and 2.0 (i.e., A260/A280 = 1.7 - 2.0).

Calculate the amount of DNA present by assuming one optical density (O.D.) unit, at 260 nm, to be equal to 50 μg/mL of double-stranded DNA (dsDNA). Use the following formula:

O.D. units X dilution factor* X 50μg/mL = amount of DNA

(*For example, if the DNA is diluted 1:10, then the dilution factor is 10.)

Note that quantitating using a UV spectrophotometer, at 260 nm, does not distinguish between single-stranded DNA, RNA, and dsDNA. A UV spectrophotometer should be used to obtain an initial estimate of genomic-DNA concentration and purity. Then, we recommend the use of PicoGreen dsDNA reagent (PicoGreen) to obtain a better estimate of concentration and purity.

Overview of the procedure for using the PUREGENE™ Genomic DNA Purification Kit on whole blood


Isolating and Quantitating DNAWorking Dilution

With PicoGreenPicoGreen from Invitrogen, Inc. is a fluorescent nucleic acid stain. Because it is specific for dsDNA, it should provide better quantity estimates then a UV spectrophotometer and also has better lower-range quantitation sensitivity.

To use PicoGreen, you must have a spectrofluorometer with fluorescein excitation and emission wavelengths of 502 nm and 523 nm, respectively.

To order PicoGreen or for more information, visit Invitrogen’s Web site (www.invitrogen.com) or contact Invitrogen at (541) 465-8300 in the United States.

Note: Quantitation may be done with a UV spectrophotometer alone. However, we recommend the use of PicoGreen dsDNA reagent, in addition to the UV spectrophotometer, for more accurate concentrations of DNA for use with the MassARRAY system.

Warning: Invitrogen, Inc. does not have data about the mutagenicity or toxicity of PicoGreen dsDNA quantitation reagent. However, because the reagent binds to nucleic acids, it should be treated and handled as a potential mutagen. Also, the reagent is in DMSO solution, which is known to facilitate the entry of organic molecules into tissues. Use double gloves when handling PicoGreen dsDNA quantitation reagent.

WorkingDilution

After quantitation, we recommend diluting a large aliquot of the DNA to a stock concentration of 50 ng/μL. This concentration of stock can be used for genotyping studies by diluting it to working concentrations. Genotyping requires 5 - 10 ng of DNA per reaction.

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Isolating and Quantitating DNAWorking Dilution

Notes:


Chapter 3Amplifying DNAfor Genotyping

Introduction This chapter covers amplifying genomic DNA for use with the MassARRAY system, using the polymerase chain reaction (PCR). DNA must be amplified prior to using the MassARRAY system.

This manual covers reactions for multiple plexing levels which have been broken down into two general ranges; 1-18 and 19-36+ plex levels. Post PCR reactions have been standardized for these two general plex ranges and can be found in Chapters 4 through 7. However, the PCR reactions should be viewed as more flexible in terms of dNTP concentrations and Taq amounts. For example, a successful uni-plex reaction can consist of 50 uM dNTPs and 0.1 U of Taq, for a successful 12 plex you would use 200 uM dNTPs and 0.5 U of Taq, and for a successful 36 plex you would use 500 uM dNTPs and 1 U of Taq. The PCR coctail tables outlined in this chapter are designed for moderate plex levels such as a 24 plex. It is recommended that you adjust your PCR cocktails according to your assay plex level.

Important: The polymerase chain reaction (PCR) is a patented process. You must have a license to perform PCR amplification. Purchase of MassARRAY system components or iPLEX Gold reagents does not confer a license to perform PCR. Information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501.

The sections in this chapter contain recommended protocols for amplifying DNA using PCR.

Note: This chapter contains recommended protocols. SEQUENOM does not guarantee these protocols to improve results. If you have amplification protocols in place, use them—note that you must ultimately have 5 μLof amplification product for each sample.

Designing PCRPrimers

When designing PCR primers, it is important to consider primer dimer formation. This is particularly important when designing primers for multiplexed reactions. SEQUENOM’s Assay Design software checks primer designs for possible dimer formation. If you are not able to use Assay Design, it is recommended you use some other program that checks for possible primer dimers.


Amplifying DNA for Genotyping384 Reactions

384 Reactions There are two recommended protocols for 384 PCR reactions, covering these alternative pipetting schemes:

• Same multiplexed assays for all wells, different DNA in the wells.• Same DNA in all wells, different multiplexed assays for the wells.

To perform 384 PCR reactions (same multiplexed assays, different DNA)

Note: These instructions cover performing PCR for a whole 384-well microtiter plate of reactions in which the same assay will be applied to different DNA.

1. Prepare a PCR cocktail as described in the following table.

Note: Add the reagents in the order in which they appear in the table.

Note: DNA (5 - 10 ng/μL) will be added in the next step.

2. To each well of a 384-well microtiter plate (Marsh Biomedical Products, Inc. #SP 0401 Sequen), add 1 μL of the appropriate genomic DNA (5-10 ng/μL).

3. Dispense 4 μL of the PCR cocktail into each well of the 384-well plate.

4. Centrifuge the microtiter plate at 1000 RPM for 1 minute.

5. Gently mix or vortex the plate, and spin down before thermocycling.

Table 3: Multiplexed PCR cocktail, without DNA, for 384 reactions (same multiplexed assays, different DNA)

Reagent Conc. in 5μL Volume (1rxn)

Volume (384rxns)*

Water (HPLC grade) NA 1.850 μL 980.38 μL

PCR Buffer (10x) (15 mM MgCl2) 1.25x 0.625 μL 331.3 μL

MgCl2 (25 mM) ** 1.625 mM 0.325 μL 172.3 μL

dNTP mix (25 mM each) *** 500 μM 0.100 μL 53.0 μL

Primer mix (500 nM each) 100 nM 1.000 μL 530.0 μL

HotstarTaq® (5 U/μL)**** 0.5 U/rxn 0.100 μL 53.0 μL

Total Volume: 4.000 μL 2120.0 μL

*Volumes include a 38% overhang**The final MgCl2 concentration is 3.5 mM, 1.875 mM from the PCR buffer and 1.625 mM from the MgCl2***No more than 5 freeze-thaw cycles **** For plexes >27, increase HotstarTaq® to 1U/rxn



6. Thermocycle the 384-well microtiter plate as follows:

To perform 384 PCR reactions (different multiplexed assays, same DNA)

Note: These instructions cover performing PCR for a whole 384-well microtiter plate of reactions in which different assays will be applied to the same DNA.

Note: The current configuration of the liquid handler allows a maximum of 96 different assays to be applied to a microtiter plate. For a 384-well microtiter plate of reactions, each assay is applied to four wells.

1. Prepare a PCR cocktail as described in the following table.


Note: Primer mix (500 nM each) will be added in the next step.

2. To each well of a 384-well microtiter plate (Marsh Biomedical Products, Inc. #SP 0401 Sequen), add 1 μL of the appropriate primer mix (500 nM each).

Note: For multiplexed assays, the PCR primers must have the 10-mer tag: (5'-ACGTTGGATG-3’).

3. Dispense 4 μL of the PCR cocktail into each well of the 384-well plate.

4. Gently mix or vortex the plate, and spin down before thermocycling.

Table 4: Multiplexed PCR cocktail, without primers, for 384 reactions (different multiplexed assays, same DNA)

Reagent Conc. in 5μL Volume (1rxn)

Volume (384rxns)*

Water (HPLC grade) NA 1.850 μL 980.38μL

PCR Buffer (10x) (15 mM MgCl2) 1.25x 0.625 μL 331.3 μL

MgCl2 (25 mM) ** 1.625 mM 0.325 μL 172.3 μL

dNTP mix (25 mM each) *** 500 μM 0.100 μL 53.0 μL

Genomic DNA (5-10 ng/μL) 5-10 ng/rxn 1.000 μL 530.0 μL

HotstarTaq® (5 U/μL) 0.5 U/rxn 0.100 μL 53.0 μL


*Volumes include a 38% overhang**The final MgCl2 concentration is 3.5 mM, 1.875 mM from the PCR buffer and 1.625 mM from the MgCl2***No more than 5 freeze-thaw cycles **** For plexes >27, increase HotstarTaq® to 1U/rxn

94° C for 15 minutes94° C for 20 seconds56° C for 30 seconds72° C for 1 minute72° C for 3 minutes4° C forever

45 cycles



5. Centrifuge the microtiter plate at 1000 RPM for 1 minute.

6. Thermocycle the 384-well microtiter plate as follows:

94° C for 15 minutes94° C for 20 seconds56° C for 30 seconds72° C for 1 minute72° C for 3 minutes4° C forever

45 cycles



Notes:




Chapter 4Processing Low Plex iPLEX Gold

Reactions for One Destination Plate(1plex - 18plex)

Introduction This chapter covers processing of low plex iPLEX Gold genotyping reactions for low plexing levels (1 - 18) on the liquid handler. The instructions and volumes presented in this chapter cover liquid handling from one source plate to one destination plate. If you prefer to dispense from one source plate to two destination plates, see “Chapter 6 Processing Low iPLEX Gold Reactions for Two Destination Plates” on page 57.

Note: The term sample microtiter plate refers to the 384-well microtiter plate of amplification products (“sample”) on which you want to perform the low plex iPLEX Gold reaction.

The main steps in processing are:

Prepare and add shrimp alkaline phosphatase (SAP) to the amplification product and then incubate

Adjust extension primers, prepare, and add the low plex iPLEX Gold reaction cocktail

Thermocycle the low plex iPLEX Gold reaction

Clean up the low plex iPLEX Gold reaction product

Detailed instructions for each of these main steps are provided in this chapter.

Note: For Multimek users, substitute the following new methods wherever appropriate:

SAP ADDITION V3.0 (384HMEAB.MTH)COCKTAIL ADDITION V3.0 (384HMEBB.MTH)WATER ADDITION v2.0 (384HMEDA.MTH)

Contact Sequenom Customer Support for iPLEX Gold upgrade if version numbers differ from above.

Source PlateThe microtiter plate from which liquid is aspirated.

Destination PlateThe microtiter plate to which liquid is dispensed. 1

2

34


Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)Daily and Weekly Maintenance

Daily andWeekly

Maintenance

For complete instructions on maintenance tasks, see the MassARRAY Liquid Handler User’s Guide. Maintenance methods are located in the Maintenance Methods folder within the Matrix ControlMate software.

Note: When opening a method, choose Cancel in the File Locked dialog box.

Daily TasksEach day, before you use the liquid handler to process an low plex iPLEX Gold reaction, you should:

1. Check the wash system tanks. Fill the supply tank and empty the waste tank if necessary.

2. Make sure the wash station is on deck position 2.

3. Run the Liquid Handler Startup Routine method.

4. At the end of each day, run the Liquid Handler Shutdown Routine method.

Weekly Tasks

Note: If instrument is in daily use, this maintenance can be performed bimonthly.

At the end of each week, after you have completed processing reactions and have run the Liquid Handler Shutdown Routine method, you should:

• Run the Liquid Handler Weekly Maintenance Routine method. • Wipe down the instrument, deck, and work area with water or mild detergent.


Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)Preparing and Adding SAP

Preparing andAdding SAP

The first step in processing the low plex iPLEX Gold reaction is to prepare and add SAP to amplification products and then incubate.

To prepare the SAP enzyme solution:1. In a 1.5 mL tube, prepare the SAP enzyme solution as described in the following

table.


2. Hold the 1.5 mL tube, containing the SAP enzyme solution, to a vortex for five seconds to mix the solution.

3. Centrifuge the 1.5 mL tube of SAP enzyme solution for ten seconds at 5000 RPM.

4. In a new 96-well, polystyrene microtiter plate (96-Well Plate Vee Bottom, Sarstedt, Inc. #82.1583), pipette 85 μL of SAP enzyme solution into each well of row H. See the following illustration.

Note: The wells in row H will be used as reservoirs from which you will distribute SAP enzyme solution to the rest of the wells in the microtiter plate.

1

Table 5: SAP Enzyme Solution

Reagent Volume (1rxn) Volume (384rxns)**

Water (HPLC grade) 1.53 μL 810.9 μL

SAP Buffer (10x) 0.17 μL 90.1 μL

SAP enzyme (1.7 U/μL) 0.30 μL 159.0 μL

Total Volume 2.00 μL 1060.0 μL

** Volumes include a 38% overhang

A

H

B

C

D

E

F

G

1 2 3 4 5 6 7 8 9 10 11 12

Dispense 85 μL of SAP enzyme solution into each well of this rowDiagram of a 96-well polystyrene microtiter

plate (Sarstedt #82.1583)



5. Using a twelve-channel pipettor, draw from the wells in row H and distribute 10 μL to each well in rows A-G. See the following illustration.

Note: The SAP enzyme solution is moderately viscous. Use care when pipetting to minimize loss of solution due to adhesion to the pipettor tips.

Caution: When pipetting, be sure to pipette into the centers of microtiter plate wells—droplets must not be placed so they adhere to well-walls. Also, make sure there are no air bubbles in the wells. Centrifuge a microtiter plate at 1600 RPM for one minute to remove air bubbles and collect liquid at the center of wells.

When you are done distributing the SAP enzyme solution, each well in rows A-G should have 10 μL of the SAP enzyme solution. The wells in row H should have 15 μL.

Note: These volumes are approximate; actual volumes may be slightly less due to pipetting loss.

Note: The 38% overhang in step 1 can be reduced to 30% when a single pipette repeater is used to fill the 96-well Vee Bottom plate.

6. Proceed to the following section, “To add the SAP enzyme solution.”

H

D

E

F

G

Diagram of a 96-well polystyrene microtiter plate (partial view; Sarstedt #82.1583)

10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

Drawing from the wells in row H, distribute10 μL of SAP enzyme solution into each of the other wells



To add the SAP enzyme solution:1. Place the 96-well microtiter plate of the SAP solution on position 1 of the liquid-

handler deck.

2. If the 384-well microtiter sample plate is sealed, remove the plate seal.

3. Centrifuge the 384-well microtiter sample plate containing amplification products at 1000 RPM for 1 minute.

4. Place the 384-well microtiter sample plate on a plate flattener. Then, place it on position 3 of the liquid-handler deck.

1 2

3 4

Wash StationSAP

Diagram of the liquid-handler deck with 96-well SAP plate added to position 1

1 2

3 4

Wash Station

Sample

384-well plates must be on plate flatteners

SAP

Diagram of the liquid-handler deck with 384-well sample microtiter plate added to position 3



Caution: If you must transfer the amplification products from the original microtiter plate to another microtiter plate, transfer it to a microtiter plate that has been “shrunken” by thermocycling. Do not transfer it to a non-thermocycled microtiter plate.

5. On the liquid handler controller PC, open the 384 Methods folder and the iPLEX-hME Methods folder. Then, run the SAP Addition (96 to 384) method.

6. When prompted for How Many 384 Plates, leave the default setting unchanged. The default setting is for one destination plate.

By default, 1 is entered for the number of plates.

2 μL of SAP enzyme solution is added to each well in the 384-well sample microtiter plate.

7. When the method is complete, remove and discard the 96-well microtiter plate of SAP solution.

To incubate the SAP enzyme solution:1. Remove the 384-well sample microtiter plate and flattener from position 3.

2. Seal the 384-well sample microtiter plate with plate sealing film. Then, remove the microtiter plate from its plate flattener.

Make sure the edges of the plate sealing film are well-sealed.

3. Centrifuge the 384-well sample microtiter plate at 1000 RPM for 1 minute.

Edit Global Values dialog box

Diagram of the liquid-handler deck configured for SAP addition1 2

3 4

Wash Station

Sample


SAP

Remove and discard the 96-well SAP plate



4. Incubate the 384-well sample microtiter plate as follows:

1. 37º C for 40 minutes.2. 85º C for 5 minutes.3. 4º C forever.

Note: If you have a programmable thermal cycler, enter the preceding program and name it SAP. When processing other low plex iPLEX Gold reactions in the future, simply run the SAP program for this step.

5. While the sample microtiter plate is thermocycling, begin preparing the low plex iPLEX Gold reaction cocktail. Proceed to “Adjusting, Preparing, and Adding the Low Plex iPLEX Gold Reaction Cocktail” on the next page.


Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)Adjusting, Preparing, and Adding the Low Plex iPLEX Gold Reaction Cocktail

Adjusting,Preparing, and

Adding the LowPlex iPLEX

Gold ReactionCocktail

After SAP enzyme has been added to the amplification product (see the preceding section), the next step is to adjust the extension primers and then prepare and add the low plex iPLEX Gold reaction cocktail.

To adjust the extension primersWhen conducting multiplexing experiments, adjusting the concentrations of oligos to equilibrate signal-to-noise ratios is required. A general method to adjust extension primers is to divide the primers into a low mass group and a high mass group. All primers in the high mass group are doubled in concentration with respect to the low mass group. For example, in a 24-plex, the 12 lowest mass primers would be at a concentration of 0.625 μM and the 12 highest mass primers would be at 1.25 μM in the final 9 μL reaction. More detailed protocols for extension primer adjustment are described in Appendix A “Extension Primer Adjustment Methods” on page 93. See also the “Extension Primer Adjustment” section of the “Plate Editor” chapter found in the iPLEX Software Guide.

To prepare the Low Plex iPLEX Gold reaction cocktail (same multiplexed assays, different DNAs for 96 to 384 dispensing)1. In a 1.5 mL tube, prepare the low plex iPLEX Gold reaction cocktail as described

in the following table.


Note: If primer extension rate appears low for 25% or more of your assays increase termination mix and termination enzyme to 1X concentration.

2

Table 6: Multiplexed low plex iPLEX Gold reaction cocktail (same multiplexed assays, different DNAs)

Reagent Conc. in 9 μL Volume (1rxn)

Volume (384rxns)**


iPLEX Buffer Plus (10x) 0.222X 0.200 μL 106.00 μL

iPLEX Termination mix 0.5x 0.100 μL 53.00 μL

Primer mix (7 μM: 14 μM)* 0.625 μM: 1.25 μM 0.94μL 498.12 μL

iPLEX enzyme 0.5x 0.0205 μL 10.87 μL


* 7 μM and 14 μM illustrate the doubled concentration of the high mass primers. Low mass primers should be at 0.625 uM and high mass primers at 1.25 uM in the final 9 μL reaction. See Appendix A “Extension Primer Adjustment Methods” on page 93 for more information.




2. Into a new, 96-well microtiter plate (Sarstedt #82.1583 96-Well Plate Vee Bottom), dispense 85 μL of low plex iPLEX Gold reaction cocktail into each well of row H. See the following illustration.

Note: The wells in row H will be used as reservoirs from which you will distribute low plex iPLEX Gold reaction cocktail into the rest of the wells.



When you are done distributing the low plex iPLEX Gold reaction cocktail, each well in rows A-G should have 10 μL of the low plex iPLEX Gold reaction cocktail. The wells in row H should have 15 μL.


A

H

B

C

D

E

F

G

1 2 3 4 5 6 7 8 9 10 11 12

Dispense 85 μL of low plex iPLEX Gold reaction cocktail into each well of this rowDiagram of a 96-well polystyrene microtiter


H

D

E

F

G



Drawing from the wells in row H, distribute10 μL of low plex iPLEX Gold reaction cocktail into each of the other wells




4. Centrifuge the cocktail microtiter plate at 1600 RPM for one minute.

5. Proceed to “To add the low plex iPLEX Gold reaction cocktail (96 to 384 dispensing)” on page 27.

To prepare the Low Plex iPLEX Gold reaction cocktail (same DNA, different multiplexed assays for 96 to 384 dispensing)1. In a 1.5 mL tube, prepare the low plex iPLEX Gold reaction cocktail, without

primer mix, as described in the following table.


Note: Primer mix (7 uM: 14 uM) will be added in the next step. 7 mM and 14 mM illustrate the doubled concentration of the high mass primers. Low mass primers should be at 0.625 uM and high mass primers at 1.25 uM in the final 9 µL reaction.


2. To each well of a 96-well microtiter plate (96-Well Plate Vee Bottom, Sarstedt, Inc. #82.1583), add 4 µL of the appropriate primer mix (7 uM: 14 uM).

3. Distribute 6 µL of reaction cocktail to each well of the 96-well plate.

At this point, you should have one 96-well microtiter plate of reaction cocktail.


5. Proceed to “To add the low plex iPLEX Gold reaction cocktail (96 to 384 dispensing)” on the next page.

Table 7: Multiplexed low plex iPLEX Gold reaction cocktail (same DNA, different assays)


Volume (384rxns)*






* Volumes include a 38% overhang



To add the low plex iPLEX Gold reaction cocktail (96 to 384 dispensing)1. Place the 96-well cocktail microtiter plate onto position 1 of the liquid-handler

deck.

2. Remove the 384-well sample microtiter plate from the thermal cycler and centrifuge at 1000 RMP for one minute. When complete, remove the plate sealing film.

3. Place the 384-well sample microtiter plate on a plate flattener. Then, place it on deck position 3.

4. On the liquid handler controller PC, open the 384 Methods folder and the iPLEX-hME Methods folder. Then, run the Cocktail Addition (96 to 384) method.

5. When prompted for How Many 384 Plates, leave the default setting unchanged. The default setting is for 1 destination plate.

2 μL of low plex iPLEX Gold reaction cocktail is added to each well of the sample microtiter plate.

1 2

3 4

Wash StationCocktail

Diagram of the liquid-handler deck with 96-well V-bottom cocktail plate added to position 1

1 2

3 4


Sample

Wash Station

Sample


Cocktail




iPLEX™

Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)Thermocycling the Low Plex iPLEX Gold Reaction

Thermocyclingthe Low PlexiPLEX Gold

Reaction

After you have added the low plex iPLEX Gold reaction cocktail to the 384-well sample microtiter plate (see the preceding section), the next step is to process the low plex iPLEX Gold reaction by thermocycling.

To thermocycle the low plex iPLEX Gold reaction1. Remove the 384-well sample microtiter plate and flattener from position 3 on the

liquid handler deck.



3. Centrifuge the 384-well sample microtiter plate at 1000 RPM for one minute.

4. Thermocycle the 384-well sample microtiter plate as follows:

For example, an MJ Thermocycler would be programmed as follows:

I. 94º C for 30 secondsII. 94º C for 5 seconds III. 52º C for 5 secondsIV. 80º C for 5 secondsV. GOTO III, 4 more times VI. GOTO II, 39 more timesVII. 72º C for 3 minutesVII. 4º C forever

The 200-short-cycle program uses two cycling loops, one of five cycles that sits inside a loop of 40 cycles. These two loops result in a 200-cycle program. The sample is denatured at 94º C. Strands are annealed at 52º C for 5 seconds and extended at 80º C for 5 seconds. The annealing and extension cycle is repeated four more times for a total of five cycles and then looped back to a 94º C denaturing step for 5 seconds and then enters the 5 cycle annealing and extension loop again. The five annealing and extension steps with the single denaturing step are repeated an additional 39 times for a total of 40. The 40 cycles of the 5 cycle annealing and extension steps equate to a total of 200 cycles (5x40). A final extension is done at 72º C for three minutes and then the sample is cooled to 4º C.

3

5 cycles 40 cycles

• 94º C for 30 seconds• 94º C for 5 seconds• 52º C for 5 seconds• 80º C for 5 seconds• 72º C for 3 minutes• 4º C forever


Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)Thermocycling the Low Plex iPLEX Gold Reaction

Note: If you have a programmable thermal cycler, enter the preceding program and name it iPLEX Gold Extend. When processing other iPLEX Gold reactions in the future, simply run the iPLEX Gold Extend program for this step.

5. Proceed to “Cleaning Up the Low Plex iPLEX Gold Reaction Products” on the next page..

Note: The cleanup of low plex iPLEX Gold reaction products involves adding water and then Clean Resin to the sample microtiter plate. If you are not ready to add Clean Resin, you may store the sample microtiter plate. Seal the microtiter plate with plate-sealing film and store it at -20º C.

Caution: When you are ready to add Clean Resin, let the microtiter plate thaw to room temperature before adding the resin.


Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)Cleaning Up the Low Plex iPLEX Gold Reaction Products

Cleaning Upthe Low Plex

iPLEX GoldReactionProducts

After the low plex iPLEX Gold reaction has been processed by thermocycling (see the preceding section), the next step is to clean up the low plex iPLEX Gold reaction products with Clean Resin (resin). This cleanup step is important to optimize mass spectrometry analysis of the low plex iPLEX Gold reaction products.

Quick overview of the process:

• Spread Clean Resin onto the 384-well dimple plate.• Add nanopure water to each well of the 384-well sample microtiter plate.• Add Clean Resin to the 384-well sample microtiter plate.• Rotate and centrifuge the 384-well sample microtiter plate.

See the instructions below.

Important: You must wear gloves and safety glasses when handling all equipment, components, and reagents such as Clean Resin.

Also, when not in use, keep the container of Clean Resin tightly closed. This is necessary to prevent the drying out of the resin.

To prepare a plate of resin

Note: Perform these steps on a clean plastic sheet. The excess resin that is scraped off the dimple plate will fall to the plastic sheet. You can return the excess resin to its container for future use.

1. Using the elongated spoon, transfer resin from its container onto the 6 mg resin 384-well dimple plate.

Important: Be sure to use the 384-well plate with 6mg dimple reservoirs. See the illustration to the right.

2. Use the scraper to spread resin into the wells of the dimple plate.

4

Elongated SpoonThis is a plastic spoon with an elongated handle (Part Number 11217). Use it to scoop resin out of its container.

384-Well dimple plate:384 wells arranged in a 16 X 24 gridDimple Plate

Use the 3mg dimple plate twice, or use a 6mg dimple plate (Part Number 11235).

ScraperThe scraper is a flat, piece of plastic (Part Number 11212). It fits the dimensions of the dimple plates and is used to remove excess resin.

Note: Make sure there is resin in each well

Resin

Scraper

Sweep the scraper back and forth across the dimple plate to spread the resin



3. Scrape excess resin off the dimple plate using the scraper.

Return the excess resin to its container.

4. Let the resin stand in the dimple plate for at least 20 minutes.

While letting the resin stand in the dimple plate, add water to the sample microtiter plate. See the next set of steps.

To add water to the 384-well sample microtiter plate1. Place a reservoir of 80 mL of nanopure water on position 1 of the liquid-handler

deck.


3. When the 384-well microtiter plate is done centrifuging, remove it from the thermal cycler and remove the plate sealing film.

Scrape away from the small, rounded post

1 2

3 4

Wash StationWater Reservoir

Diagram of the liquid-handler deck with water reservoir addeto position 1



4. Place the 384-well sample microtiter plate on a plate flattener. Then, place it on position 3 of the liquid-handler deck.

5. On the liquid handler controller PC, open the 384 Methods folder and the iPLEX-hME Methods folder. Then, run the 16 µL Water Addition (384) method.


16 µL of water is added to the sample microtiter plate.

7. Remove the 384-well sample microtiter plate and flattener from position 3.



9. Centrifuge the microtiter plate (for 30 seconds at 1600 RPM, or until the air bubbles are gone).

10. Proceed to “To add resin to the sample microtiter plate” below.

To add resin to the sample microtiter plate1. Gently place the sample microtiter plate, upside-down, onto the dimple plate.

1 2

3 4


Sample

Wash Station

Sample


Water Reservoir

Diagram of the liquid-handler deck with sample microtiter platadded to position 3

Dimple plate

384-well sample microtiter plate (upside-down)

Make sure the microtiter plate rests against this small, rounded post on the dimple plate; this aligns the wells in the microtiter plate with the wells in the dimple plate



2. Holding the sample microtiter plate and the dimple plate together, gently flip them over so the resin falls out of the dimple plate into the wells of the microtiter plate.

Tap the dimple plate so the resin falls out into the microtiter plate. Make sure all the resin in the dimple plate falls out into the microtiter plate wells.

3. Proceed to “To rotate and centrifuge the low plex iPLEX Gold reaction products” below.

Note: Make sure there are no air bubbles in the wells before rotating the plate. If there are air bubbles, centrifuge the plate briefly, and then rotate it.

To rotate and centrifuge the low plex iPLEX Gold reaction products1. Rotate the sample microtiter plate on a rotator for five minutes, at room

temperature.

The rotator must rotate the microtiter plate 360º about its long axis.

2. Centrifuge the sample microtiter plate at 3200 g for five minutes.

3. The low plex iPLEX Gold reaction products are now ready for transfer to a SpectroCHIP, using the MassARRAY Nanodispenser.

Note: If you are not ready to transfer the low plex iPLEX Gold reaction products to a SpectroCHIP, you can store the microtiter plate at -20º C until you are ready. Place adhesive sealing foil (Marsh Biomedical Products, Inc. #AB-0626) on the microtiter plate before storing. Make sure the edges of the sealing foil are well-sealed. Do not store the microtiter plate for more than two weeks.If you have stored microtiter plate(s) of low plex iPLEX Gold reaction products, thaw, rotate, and then centrifuge the microtiter plate(s) (at 3200 g for five minutes) before transferring the reaction products to a SpectroCHIP.

384-well microtiter plate and dimple plate

Flip them over so the dimple plate is on top


Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)Nanodispensing

Nanodispensing Once liquid handling is complete, perform nanodispensing of low plex iPLEX Gold reaction products onto a SpectroCHIP. For instructions, see the MassARRAY Nanodispenser User’s Guide (Samsung).

DefiningAssays and

Plates

Once nanodispensing is complete, define how assays and plates are to be set up in the MassARRAY database. For instructions, see the “Defining Assays” and “Defining Plates” chapters in the MassARRAY Typer Software User’s Guide.

AcquiringSpectra

After you have defined assays and plates, acquire spectra using the MassARRAY Analyzer Compact mass spectrometer. For instructions, see the “Acquiring Spectra” chapter in the MassARRAY Analyzer Compact User’s Guide.


Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)Acquiring Spectra

Notes:


Chapter 5Processing High Plex iPLEX Gold

Reactions for One Destination Plate(19plex - 36+plex)

Introduction This chapter covers processing of high plex iPLEX Gold genotyping reactions for high plexing levels (19 - 36+) on the liquid handler. The instructions and volumes presented in this chapter cover liquid handling from one source plate to one destination plate. If you prefer to dispense from one source plate to two destination plates, see “Chapter 7 Processing high plex iPLEX Gold Reactions for Two Destination Plates” on page 75.

Note: The term sample microtiter plate refers to the 384-well microtiter plate of amplification products (“sample”) on which you want to perform the high plex iPLEX Gold reaction.



Adjust extension primers, prepare, and add the high plex iPLEX Gold reaction cocktail

Thermocycle the high plex iPLEX Gold reaction

Clean up the high plex iPLEX Gold reaction product






Destination PlateThe microtiter plate to which liquid is dispensed. 1

2

34


Processing High Plex iPLEX Gold Reactions for One Destination Plate (19plex - 36+plex)Daily and Weekly Maintenance

Daily andWeekly

Maintenance



Daily TasksEach day, before you use the liquid handler to process an high plex iPLEX Gold reaction, you should:





Weekly Tasks





Processing High Plex iPLEX Gold Reactions for One Destination Plate (19plex - 36+plex)

Preparing andAdding SAP

The first step in processing the high plex iPLEX Gold reaction is to prepare and add SAP to amplification products and then incubate.

To prepare the SAP enzyme solution:1. In a 1.5 mL tube, prepare the SAP enzyme solution as described in the following

table.


2. Hold the 1.5 mL tube, containing the SAP enzyme solution, to a vortex for five seconds to mix the solution.

3. Centrifuge the 1.5 mL tube of SAP enzyme solution for ten seconds at 5000 RPM.


Note: The wells in row H will be used as reservoirs from which you will distribute SAP enzyme solution to the rest of the wells in the microtiter plate.

1








A

H

B

C

D

E

F

G

1 2 3 4 5 6 7 8 9 10 11 12




Processing High Plex iPLEX Gold Reactions for One Destination Plate (19plex - 36+plex)Preparing and Adding SAP







6. Proceed to the following section, “To add the SAP enzyme solution.”

H

D

E

F

G







handler deck.



4. Place the 384-well microtiter sample plate on a plate flattener. Then, place it on position 3 of the liquid-handler deck.

1 2

3 4

Wash StationSAP


1 2

3 4

Wash Station

Sample


SAP



Processing High Plex iPLEX Gold Reactions for One Destination Plate (19plex - 36+plex)Preparing and Adding SAP



6. When prompted for How Many 384 Plates, leave the default setting unchanged. The default setting is for one destination plate.

By default, 1 is entered for the number of plates.

2 μL of SAP enzyme solution is added to each well in the 384-well sample microtiter plate.


To incubate the SAP enzyme solution:1. Remove the 384-well sample microtiter plate and flattener from position 3.



3. Centrifuge the 384-well sample microtiter plate at 1000 RPM for 1 minute.


Diagram of the liquid-handler deck configured for SAP addition1 2

3 4

Wash Station

Sample


SAP

Remove and discard the 96-well SAP plate



4. Incubate the 384-well sample microtiter plate as follows:

1. 37º C for 40 minutes.2. 85º C for 5 minutes.3. 4º C forever.

Note: If you have a programmable thermal cycler, enter the preceding program and name it SAP. When processing other high plex iPLEX Gold reactions in the future, simply run the SAP program for this step.

5. While the sample microtiter plate is thermocycling, begin preparing the iPLEX Gold reaction cocktail. Proceed to “Adjusting, Preparing, and Adding the High Plex iPLEX Gold Reaction Cocktail” on the next page.


Processing High Plex iPLEX Gold Reactions for One Destination Plate (19plex - 36+plex)Adjusting, Preparing, and Adding the High Plex iPLEX Gold Reaction Cocktail


Adding theHigh Plex

iPLEX GoldReactionCocktail

After SAP enzyme has been added to the amplification product (see the preceding section), the next step is to adjust the extension primers and then prepare and add the high plex iPLEX Gold reaction cocktail.

To adjust the extension primersWhen conducting high multiplexing experiments, adjusting the concentrations of oligos to equilibrate signal-to-noise ratios is required. A general method to adjust high plex extension primers is to divide the primers into a low mass group, a medium mass group, and a high mass group. All primers are adjusted in a three tier fashion, one tier for every twelve primers. For example, in a 36-plex, the 12 lowest mass primers would be at a concentration of 0.0.84 μM, the 12 middle mass primers would be at a concentration of 1.04 μM and the 12 highest mass primers would be at 1.25 μM in the final 9 μL reaction. More detailed protocols for extension primer adjustment are described in Appendix A “Extension Primer Adjustment Methods” on page 93. See also the “Extension Primer Adjustment” section of the “Plate Editor” chapter found in the iPLEX Software Guide.

To prepare the High Plex iPLEX Gold reaction cocktail (same multiplexed assays, different DNAs for 96 to 384 dispensing)1. In a 1.5 mL tube, prepare the high plex iPLEX Gold reaction cocktail as described



2

Table 9: Multiplexed high plex iPLEX Gold reaction cocktail (same multiplexed assays, different DNAs)


Volume (384rxns)**



iPLEX Termination mix 1x 0.200 μL 106.00 μL

Primer mix (8 μM: 10 μM: 12 μM)* 0.84μM:1.04μM: 1.25 μM

0.940μL 498.20 μL

iPLEX enzyme 1x 0.041 μL 21.73 μL


* 8 μM, 10 μM and 12 μM illustrate the three tiered concentration of the low to high mass primers. Low mass primers should be at 0.84 uM, medium mass primers should be 1.04 μM and high mass primers at 1.25 μM in the final 9 μL reaction. See Appendix A “Extension Primer Adjustment Methods” on page 93 for more information.




2. Into a new, 96-well microtiter plate (Sarstedt #82.1583 96-Well Plate Vee Bottom), dispense 85 μL of high plex iPLEX Gold reaction cocktail into each well of row H. See the following illustration.

Note: The wells in row H will be used as reservoirs from which you will distribute high plex iPLEX Gold reaction cocktail into the rest of the wells.



When you are done distributing the high plex iPLEX Gold reaction cocktail, each well in rows A-G should have 10 μL of the high plex iPLEX Gold reaction cocktail. The wells in row H should have 15 μL.


A

H

B

C

D

E

F

G

1 2 3 4 5 6 7 8 9 10 11 12

Dispense 85 μL of high plex iPLEX Gold reaction cocktail into each well of this rowDiagram of a 96-well polystyrene microtiter


H

D

E

F

G



Drawing from the wells in row H, distribute10 μL of high plex iPLEX Gold reaction cocktail into each of the other wells





5. Proceed to “To add the High Plex iPLEX Gold reaction cocktail (96 to 384 dispensing)” on page 47.

To prepare the High Plex iPLEX Gold reaction cocktail (same DNA, different multiplexed assays for 96 to 384 dispensing)1. In a 1.5 mL tube, prepare the high plex iPLEX Gold reaction cocktail, without



Note: Primer mix (8 μM: 10 μM: 12 μM) will be added in the next step. 8 μM, 10 μM and 12 μM illustrate the three tiered concentration of the low to high mass primers. Low mass primers should be at 0.84 uM, medium mass primers should be 1.04 μM and high mass.

2. To each well of a 96-well microtiter plate (96-Well Plate Vee Bottom, Sarstedt, Inc. #82.1583), add 4 µL of the appropriate primer mix (8 μM 10 μM 12 μM).Distribute 6 µL of reaction cocktail to each well of the 96-well plate.



4. Proceed to “To add the High Plex iPLEX Gold reaction cocktail (96 to 384 dispensing)” on the next page.

Table 10: Multiplexed high plex iPLEX Gold reaction cocktail (same DNA, different assays)


Volume (384rxns)*









To add the High Plex iPLEX Gold reaction cocktail (96 to 384 dispensing)1. Place the 96-well cocktail microtiter plate onto position 1 of the liquid-handler

deck.

2. Remove the 384-well sample microtiter plate from the thermal cycler and centrifuge at 1000 RMP for one minute. When complete, remove the plate sealing film.

3. Place the 384-well sample microtiter plate on a plate flattener. Then, place it on deck position 3.



2 μL of high plex iPLEX Gold reaction cocktail is added to each well of the sample microtiter plate.

1 2

3 4



1 2

3 4


Sample

Wash Station

Sample


Cocktail




6. When the method is complete, remove and discard the 96-well microtiter plate of high plex iPLEX Gold reaction cocktail.

7. Proceed to “Thermocycling the High Plex iPLEX Gold Reaction” on the next page.

1 2

3 4


Sample

Wash Station

Sample


Cocktail

Diagram of the liquid-handler deck configured for cocktail addition

Remove and discard the 96-well cocktail plate



After you have added the high plex iPLEX Gold reaction cocktail to the 384-well sample microtiter plate (see the preceding section), the next step is to process the high plex iPLEX

November 10, 2006 49 iPLEX™ Gold Application GuideDoc. 11555 R00, CO 060190 (Matrix, Samsung, Compact)Thermocycling

the High PlexiPLEX Gold

Reaction


Cleaning Upthe High Plex


After the high plex iPLEX Gold reaction has been processed by thermocycling (see the preceding section), the next step is to clean up the high plex iPLEX


Processing High Plex iPLEX Gold Reactions for One Destination Plate (19plex - 36+plex)Cleaning Up the High Plex iPLEX Gold Reaction Products






deck.


3. When the 384-well microtiter plate is done centrifuging, remove it from the thermal cycler and remove the plate sealing film.


1 2

3 4





4. Place the 384-well sample microtiter plate on a plate flattener. Then, place it on position 3 of the liquid-handler deck.



16 µL of water is added to the sample microtiter plate.

7. Remove the 384-well sample microtiter plate and flattener from position 3.



9. Centrifuge the microtiter plate (for 30 seconds at 1600 RPM, or until the air bubbles are gone).

10. Proceed to “To add resin to the sample microtiter plate” below.

To add resin to the sample microtiter plate1. Gently place the sample microtiter plate, upside-down, onto the dimple plate.

1 2

3 4


Sample

Wash Station

Sample


Water Reservoir

Diagram of the liquid-handler deck with sample microtiter platadded to position 3

Dimple plate




Processing High Plex iPLEX Gold Reactions for One Destination Plate (19plex - 36+plex)Cleaning Up the High Plex iPLEX Gold Reaction Products



3. Proceed to “To rotate and centrifuge the high plex iPLEX Gold reaction products” below.


To rotate and centrifuge the high plex iPLEX Gold reaction products1. Rotate the sample microtiter plate on a rotator for five minutes, at room

temperature.


2. Centrifuge the sample microtiter plate at 3200 g for five minutes.

3. The high plex iPLEX Gold reaction products are now ready for transfer to a SpectroCHIP, using the MassARRAY Nanodispenser.

Note: If you are not ready to transfer the high plex iPLEX Gold reaction products to a SpectroCHIP, you can store the microtiter plate at -20º C until you are ready. Place adhesive sealing foil (Marsh Biomedical Products, Inc. #AB-0626) on the microtiter plate before storing. Make sure the edges of the sealing foil are well-sealed. Do not store the microtiter plate for more than two weeks.If you have stored microtiter plate(s) of high plex iPLEX Gold reaction products, thaw, rotate, and then centrifuge the microtiter plate(s) (at 3200 g for five minutes) before transferring the reaction products to a SpectroCHIP.





Nanodispensing Once liquid handling is complete, perform nanodispensing of high plex iPLEX Gold reaction products onto a SpectroCHIP. For instructions, see the MassARRAY Nanodispenser User’s Guide (Samsung).

DefiningAssays and

Plates


AcquiringSpectra



Processing High Plex iPLEX Gold Reactions for One Destination Plate (19plex - 36+plex)Acquiring Spectra

Notes:


Chapter 6Processing Low iPLEX Gold

Reactions for Two DestinationPlates(1plex - 18plex)

Introduction This chapter covers liquid handling methods where one source plate is dispensed to two destination plates. This “two plate” option requires you to prepare increased reagent volumes and to select two-plate method options in the Matrix software. If you prefer to dispense only to one destination plate, see “Chapter 4 Processing Low Plex iPLEX Gold Reactions for One Destination Plate (1plex - 18plex)” on page 15.

Note: The term sample microtiter plate refers to any 384-well microtiter plate of amplification products (“sample”) on which you want to perform the iPLEX Gold reaction.



Prepare and add iPLEX Gold reaction cocktail

Thermocycle the iPLEX Gold reaction

Clean up the iPLEX Gold reaction product




After the first run of each method, exchange the destination plate and rerun.



Destination PlateThe microtiter plate to which liquid is dispensed.

1

234


Processing Low iPLEX Gold Reactions for Two Destination Plates(1plex - 18plex)Daily and Weekly Maintenance

Daily andWeekly

Maintenance



Daily TasksEach day, before you use the liquid handler to process an iPLEX Gold reaction, you should:





Weekly Tasks




iPLEX™ Gold Application Guide 58 November 10, 2006 (Matrix, Samsung, Compact) Doc. 11555 R00, CO 060190

Processing Low iPLEX Gold Reactions for Two Destination Plates(1plex - 18plex)Preparing and Adding SAP to Two Destination Plates

Preparing andAdding SAP to

TwoDestination

Plates

The first step in processing the iPLEX Gold reaction is to prepare and add SAP to amplification products and then incubate.

To prepare the SAP enzyme solution:1. In a 5 mL tube, prepare the SAP enzyme solution as described in the following

table.


2. Hold the 5 mL tube, containing the SAP enzyme solution, to a shaker for five seconds to mix the solution.

3. Centrifuge the 5 mL tube of SAP enzyme solution for ten seconds at 5000 RPM.


1








A

H

B

C

D

E

F

G

1 2 3 4 5 6 7 8 9 10 11 12



Note: The wells in row H will be used as reservoirs from which you will distribute SAP enzyme solution to the rest of the wells in the microtiter plate





Caution: When pipetting, be sure to pipette into the centers of microplate wells -- droplets must not be placed so they adhere to well-walls. Also make sure there are no air bubbles in the wells. Centrifuge a microplated at 1600 RPM for one minute to remove air bubbles and collect liquid at the center of wells.




H

D

E

F

G







handler deck.



4. Place the first 384-well microtiter sample plate of amplification products on a plate flattener. Then, place it on position 3 of the liquid-handler deck. Place the second 384-well microtiter plate of amplification products on a plate flattener, and then place it on position 4.



1 2

3 4

Wash StationSAP


1 2

3 4

Wash Station

Sample Sample

384-well plates must be on plate flatteners 384-well plates must be on plate flatteners

SAP

Diagram of the liquid-handler deck with 384-well sample microtiter plates added to position 3 and 4



6. On the liquid handler controller PC, type 2 for the number of plates.

2 μL of SAP enzyme solution is added to each well in the two 384-well sample microtiter plates.


To incubate the SAP enzyme solution:1. Remove the 384-well sample microtiter plates and plate flatteners from position 3

and 4.

2. Seal the 384-well sample microtiter plates with plate sealing film. Then, remove the microtiter plates from their plate flatteners.


3. Centrifuge the 384-well sample microtiter plates at 1000 RPM for 1 minute.

4. Incubate both 384-well sample microtiter plates as follows:

1.37º C for 40 minutes.2. 85º C for 5 minutes.3. 4º C forever.

Note: If you have a programmable thermal cycler, enter the preceding program and name it SAP. When processing other iPLEX Gold reactions in the future, simply run the SAP program for this step.

5. While the sample microtiter plates are thermocycling, begin preparing the iPLEX Gold reaction cocktail. Proceed to “Adjusting, Preparing, and Adding the Low Plex iPLEX Gold Reaction Cocktail” on the next page..


If you are dispensing to 2 sample microtiter plates, you must type 2 in the How Many 384 Plates box.


Processing Low iPLEX Gold Reactions for Two Destination Plates(1plex - 18plex)Adjusting, Preparing, and Adding the Low Plex iPLEX Gold Reaction Cocktail


Adding the LowPlex iPLEX

Gold ReactionCocktail

After SAP enzyme has been added to the amplification product (see the preceding section), the next step is to adjust the extension primers and then prepare and add the low plex iPLEX Gold reaction cocktail.

To adjust the extension primersWhen conducting multiplexing experiments, adjusting the concentrations of oligos to equilibrate signal-to-noise ratios is required. A general method to adjust extension primers is to divide the primers into a low mass group and a high mass group. All primers in the high mass group are doubled in concentration with respect to the low mass group. For example, in a 24-plex, the 12 lowest mass primers would be at a concentration of 0.625 μM and the 12 highest mass primers would be at 1.25 μM in the final 9 μL reaction. More detailed protocols for extension primer adjustment are described in Appendix A “Extension Primer Adjustment Methods” on page 93. See also the “Extension Primer Adjustment” section of the “Plate Editor” chapter found in the iPLEX Software Guide.

To prepare the low plex iPLEX Gold reaction cocktail (same multiplexed assays, different DNAs for 96 to 384 dispensing)1. In a 5 mL tube, prepare the low plex iPLEX Gold reaction cocktail as described in

the following table.

Note: Add the reagents in the order in which they appear in the table..


2

Table 12: Multiplexed low plex iPLEX Gold reaction cocktail (same multiplexed assay, different DNAs)


Volume (768rxns)**




Primer mix (7 μM: 14 μM)* 0.625 μM: 1.25 μM 0.94μL 996.24 μL



* 7μM and 14μM illustrate the doubled concentration of the high mass primers. Low mass primers should be at 0.625 uM and high mass primers at 1.25 μM in the final 9 µL reaction.




2. In a new 96-well, polystyrene microtiter plate (96-Well Plate Vee Bottom, Sarstedt, Inc. #82.1583), pipette 170 μL of low plex iPLEX Gold reaction cocktail into each well of row H. See the following illustration.

Note: The wells in row H will be used as reservoirs from which you will distribute low plex iPLEX Gold reaction cocktail to the rest of the wells in the microtiter plate.


Note: These volumes are approximate, actual volumes may be slightly less due to pipetting loss.

Caution: When pipetting, be sure to pipette into the centers of microplate wells -- droplets must not be placed so they adhere to well-walls. Also make sure there are no air bubbles in the wells. Centrifuge a microplate at 1600 RPM for one minute to remove air bubbles and collect liquid at the center of wells.

When done distributing the low plex iPLEX Gold reaction cocktail, each well in rows A-G should have 20 μL of low plex iPLEX Gold reaction cocktail. The wells in row H should have 30 μL.

A

H

B

C

D

E

F

G

1 2 3 4 5 6 7 8 9 10 11 12

Dispense 170 μL of low plex iPLEX Gold reaction cocktail into each well of this rowDiagram of a 96-well polystyrene microtiter


H

D

E

F

G



Drawing from the wells in row H, distribute20 μL of low plex iPLEX Gold reaction cocktail into each of the other wells





5. Proceed to “To add the low plex iPLEX Gold reaction cocktail (96 to 384 dispensing)” on page 66.

To prepare the low plex iPLEX Gold reaction cocktail (same DNA, different multiplexed assays for 96 to 384 dispensing)1. In a 1.5 mL tube, prepare the low plex iPLEX Gold reaction cocktail, without



Note: Primer mix (7 uM: 14 uM) will be added in the next step. 7 mM and 14 mM illustrate the doubled concentration of the high mass primers. Low mass primers should be at 0.625 uM and high mass primers at 1.25 uM in the final 9 µL reaction.






5. Proceed to “To add the low plex iPLEX Gold reaction cocktail (96 to 384 dispensing)” on the next page.

Table 13: Multiplexed low plex iPLEX Gold reaction cocktail


Volume (768rxns)*









To add the low plex iPLEX Gold reaction cocktail (96 to 384 dispensing)1. Place the 96-well cocktail microtiter plate onto position 1 of the liquid-handler

deck.

2. Remove both 384-well sample microtiter plates from the thermal cycler. Centrifuge the cocktail microtiter plate at 1000 RPM for one minute. When complete, remove the plate sealing film.

3. Place the 384-well sample microtiter plates on plate flatteners. Then, place them on deck position 3 and 4.


5. When prompted for the number of plates, type 2 in the Edit Global Values dialog box.

2 μL of low plex iPLEX Gold reaction cocktail is added to each well of the sample microtiter plates.

1 2

3 4



1 2

3 4


Sample

Wash Station

Sample Sample


Cocktail

Diagram of the liquid-handler deck with sample microtiter plates added to position 3 and 4



6. When the method is complete, remove and discard the 96-well microtiter plate of low plex iPLEX Gold reaction cocktail.

7. Proceed to “Thermocycling the low plex iPLEX Gold Reaction in Two Plates” on the next page.

1 2

3 4


Sample

Wash Station

Sample Sample


Cocktail

Diagram of the liquid-handler deck configured for cocktail addition to 2 plates



Processing Low iPLEX Gold Reactions for Two Destination Plates(1plex - 18plex)Thermocycling the low plex iPLEX Gold Reaction in Two Plates

Thermocyclingthe low plexiPLEX GoldReaction inTwo Plates

After you have added the low plex iPLEX Gold reaction cocktail to the 384-well sample microtiter plates (see the preceding section), the next step is to process the low plex iPLEX Gold reaction by thermocycling.

To thermocycle the low plex iPLEX Gold reaction1. Remove the 384-well sample microtiter plates and plate flatteners from position 3

and 4 on the liquid handler deck.



3. Centrifuge the 384-well sample microtiter plates at 1000 RPM for one minute.

4. Thermocycle the 384-well sample microtiter plates as follows:




3

5 cycles40 cycles



Processing Low iPLEX Gold Reactions for Two Destination Plates(1plex - 18plex)Thermocycling the low plex iPLEX Gold Reaction in Two Plates

Note: If you have a programmable thermal cycler, enter the preceding program and name it iPLEX Gold Extend. When processing other iPLEX reactions in the future, simply run the iPLEX Gold Extend program for this step.

5. Proceed to “Cleaning Up the Low Plex iPLEX Gold Reaction Products” on the next page..

Note: The cleanup of low plex iPLEX Gold reaction products involves adding water and then Clean Resin to the sample microtiter plate. If you are not ready to add Clean Resin, you may store the sample microtiter plates. Seal the microtiter plates with plate-sealing film and store it at -20º C.

Caution: When you are ready to add Clean Resin, let the microtiter plates thaw to room temperature before adding the resin.


Processing Low iPLEX Gold Reactions for Two Destination Plates(1plex - 18plex)Cleaning Up the Low Plex iPLEX Gold Reaction Products

Cleaning Upthe Low Plex


After the low plex iPLEX Gold reaction has been processed by thermocycling (see the preceding section), the next step is to clean up the low plex iPLEX Gold reaction products with Clean Resin (resin). This cleanup step is important to optimize mass spectrometry analysis of the low plex iPLEX Gold reaction products.


• Spread Clean Resin onto the 384-well dimple plate.• Add nanopure water to each well of the 384-well sample microtiter plates.• Add Clean Resin to the 384-well sample microtiter plates.• Rotate and centrifuge the 384-well sample microtiter plates.



Also, when not in use, keep the bottle of Clean Resin tightly closed. This is necessary to prevent the drying out of the resin.






4


384-Well dimple plate:384 wells arranged in a 16 X 24 grid

DImple PlateUse the 3mg dimple plate twice, or use a 6 mg dimple plate (Part Number 11235).



Resin

Scraper









deck.

2. When the 384-well microtiter plates are done thermocycling, centrifuge the 384-well sample microtiter plates at 1000 RPM for one minute. Remove them from the thermal cycler and remove the plate sealing film.


1 2

3 4





3. Place the 384-well sample microtiter plates on plate flatteners. Then, put them on position 3 and 4 of the liquid-handler deck.


5. When prompted for How Many 384 Plates, type 2 to indicate two destination plates have been placed on the liquid handler deck.

16 µL of water is added to the sample microtiter plates.

6. Remove the 384-well sample microtiter plates and flatteners from positions 3 and 4.

7. Seal the 384-well sample microtiter plates with plate sealing film. Then, remove the microtiter plates from their plate flattener.s


8. Centrifuge the microtiter plates (for 30 seconds at 1600 RPM, or until the air bubbles are gone).

9. Proceed to “To add resin to the sample microtiter plate” on the next page..

1 2

3 4


Sample

Wash Station

Sample Sample


Water Reservoir




To add resin to the sample microtiter plate

Note: Perform the following steps for each of the two sample microtiter plates.

1. Gently place the sample microtiter plate, upside-down, onto the dimple plate.



3. Repeat these steps for the second sample microtiter plate.

4. Once both sample microtiter plates have had resin added, proceed to “To rotate and centrifuge the low plex iPLEX Gold reaction products” below.


Dimple plate






Processing Low iPLEX Gold Reactions for Two Destination Plates(1plex - 18plex)Nanodispensing

To rotate and centrifuge the low plex iPLEX Gold reaction products1. Rotate the sample microtiter plates on a rotator for five minutes, at room

temperature.


2. Centrifuge the sample microtiter plates at 3200 g for five minutes.

3. The low plex iPLEX Gold reaction products are now ready for transfer to a SpectroCHIP, using the MassARRAY Nanodispenser.

Note: If you are not ready to transfer the low plex iPLEX Gold reaction products to a SpectroCHIP, you can store the microtiter plates at -20º C until you are ready. Place adhesive sealing foil (Marsh Biomedical Products, Inc. #AB-0626) on the microtiter plates before storing. Make sure the edges of the sealing foil are well-sealed. Do not store the microtiter plates for more than two weeks.

If you have stored microtiter plates of low plex iPLEX Gold reaction products, thaw, rotate, and then centrifuge the microtiter plates (at 3200 g for five minutes) before transferring the reaction products to a SpectroCHIP.

Nanodispensing Once liquid handling is complete, perform nanodispensing of low plex iPLEX Gold reaction products onto a SpectroCHIP. For instructions, see the MassARRAY Nanodispenser User’s Guide (Samsung).

DefiningAssays and

Plates


AcquiringSpectra



Chapter 7Processing High iPLEX Gold

Reactions for Two DestinationPlates(19plex - 36+plex)

Introduction This chapter covers liquid handling methods where one source plate is dispensed to two destination plates. This “two plate” option requires you to prepare increased reagent volumes and to select two-plate method options in the Matrix software. If you prefer to dispense only to one destination plate, see “Chapter 5 Processing High Plex iPLEX Gold Reactions for One Destination Plate” on page 37.

Note: The term sample microtiter plate refers to any 384-well microtiter plate of amplification products (“sample”) on which you want to perform the high plex iPLEX Gold reaction.



Prepare and add high plex iPLEX Gold reaction cocktail

Thermocycle the high plex iPLEX Gold reaction

Clean up the high plex iPLEX Gold reaction product




After the first run of each method, exchange the destination plate and rerun.

Contact Sequenom Customer Support for iPLEX upgrade if version numbers differ from above.


Destination PlateThe microtiter plate to which liquid is dispensed.

1

234


Processing High iPLEX Gold Reactions for Two Destination Plates(19plex - 36+plex)Daily and Weekly Maintenance

Daily andWeekly

Maintenance



Daily TasksEach day, before you use the liquid handler to process an high plex iPLEX Gold reaction, you should:





Weekly Tasks





Processing High iPLEX Gold Reactions for Two Destination Plates(19plex - 36+plex)Preparing and Adding SAP to Two Destination Plates

Preparing andAdding SAP to

TwoDestination

Plates

The first step in processing the high plex iPLEX Gold reaction is to prepare and add SAP to amplification products and then incubate.

To prepare the SAP enzyme solution:1. In a 5 mL tube, prepare the SAP enzyme solution as described in the following

table.


2. Hold the 5 mL tube, containing the SAP enzyme solution, to a shaker for five seconds to mix the solution.

3. Centrifuge the 5 mL tube of SAP enzyme solution for ten seconds at 5000 RPM.


1








A

H

B

C

D

E

F

G

1 2 3 4 5 6 7 8 9 10 11 12



Note: The wells in row H will be used as reservoirs from which you will distribute SAP enzyme solution to the rest of the wells in the microtiter plate





Caution: When pipetting, be sure to pipette into the centers of microplate wells -- droplets must not be placed so they adhere to well-walls. Also make sure there are no air bubbles in the wells. Centrifuge a microplated at 1600 RPM for one minute to remove air bubbles and collect liquid at the center of wells.




H

D

E

F

G







handler deck.



4. Place the first 384-well microtiter sample plate of amplification products on a plate flattener. Then, place it on position 3 of the liquid-handler deck. Place the second 384-well microtiter plate of amplification products on a plate flattener, and then place it on position 4.



1 2

3 4

Wash StationSAP


1 2

3 4

Wash Station

Sample Sample


SAP

Diagram of the liquid-handler deck with 384-well sample microtiter plates added to position 3 and 4



6. On the liquid handler controller PC, type 2 for the number of plates.

2 μL of SAP enzyme solution is added to each well in the two 384-well sample microtiter plates.


To incubate the SAP enzyme solution:1. Remove the 384-well sample microtiter plates and plate flatteners from position 3

and 4.



3. Centrifuge the 384-well sample microtiter plates at 1000 RPM for 1 minute.

4. Incubate both 384-well sample microtiter plates as follows:

1.37º C for 40 minutes.2. 85º C for 5 minutes.3. 4º C forever.

Note: If you have a programmable thermal cycler, enter the preceding program and name it SAP. When processing other high plex iPLEX Gold reactions in the future, simply run the SAP program for this step.

5. While the sample microtiter plates are thermocycling, begin preparing the high plex iPLEX Gold reaction cocktail. Proceed to “Adjusting, Preparing, and Adding the High Plex iPLEX Gold Reaction Cocktail” on the next page..


If you are dispensing to 2 sample microtiter plates, you must type 2 in the How Many 384 Plates box.


Processing High iPLEX Gold Reactions for Two Destination Plates(19plex - 36+plex)Adjusting, Preparing, and Adding the High Plex iPLEX Gold Reaction Cocktail


Adding theHigh Plex

iPLEX GoldReactionCocktail

After SAP enzyme has been added to the amplification product (see the preceding section), the next step is to adjust the extension primers and then prepare and add the high plex iPLEX reaction cocktail.

To adjust the extension primersWhen conducting high multiplexing experiments, adjusting the concentrations of oligos to equilibrate signal-to-noise ratios is required. A general method to adjust high plex extension primers is to divide the primers into a low mass group, a medium mass group, and a high mass group. All primers are adjusted in a three tier fashion, one tier for every twelve primers. For example, in a 36-plex, the 12 lowest mass primers would be at a concentration of 0.0.84 μM, the 12 middle mass primers would be at a concentration of 1.04 μM and the 12 highest mass primers would be at 1.25 μM in the final 9 μL reaction. More detailed protocols for extension primer adjustment are described in Appendix A “Extension Primer Adjustment Methods” on page 93. See also the “Extension Primer Adjustment” section of the “Plate Editor” chapter found in the iPLEX Software Guide.

To prepare the high plex iPLEX Gold reaction cocktail (same multiplexed assays, different DNAs for 96 to 384 dispensing)1. In a 5 mL tube, prepare the high plex iPLEX Gold reaction cocktail as described


Note: Add the reagents in the order in which they appear in the table..

2

Table 15: Multiplexed high plex iPLEX Gold reaction cocktail (same multiplexed assay, different DNAs)


Volume (768rxns)**




Primer mix (8 μM: 10 μM: 12 μM)* 0.84μM:1.04μM: 1.25 μM

0.940μL 996.40 μL



* 8 μM, 10 μM and 12 μM illustrate the three tiered concentration of the low to high mass primers. Low mass primers should be at 0.84 uM, medium mass primers should be 1.04 μM and high mass primers at 1.25 μM in the final 9 μL reaction.




2. In a new 96-well, polystyrene microtiter plate (96-Well Plate Vee Bottom, Sarstedt, Inc. #82.1583), pipette 170 μL of high plex iPLEX Gold reaction cocktail into each well of row H. See the following illustration.

Note: The wells in row H will be used as reservoirs from which you will distribute high plex iPLEX Gold reaction cocktail to the rest of the wells in the microtiter plate.


Note: These volumes are approximate, actual volumes may be slightly less due to pipetting loss.

Caution: When pipetting, be sure to pipette into the centers of microplate wells -- droplets must not be placed so they adhere to well-walls. Also make sure there are no air bubbles in the wells. Centrifuge a microplate at 1600 RPM for one minute to remove air bubbles and collect liquid at the center of wells.

When done distributing the high plex iPLEX Gold reaction cocktail, each well in rows A-G should have 20 μL of high plex iPLEX Gold reaction cocktail. The wells in row H should have 30 μL.

A

H

B

C

D

E

F

G

1 2 3 4 5 6 7 8 9 10 11 12

Dispense 170 μL of high plex iPLEX Gold reaction cocktail into each well of this rowDiagram of a 96-well polystyrene microtiter


H

D

E

F

G



Drawing from the wells in row H, distribute20 μL of high plex iPLEX Gold reaction cocktail into each of the other wells





5. Proceed to “To add the high plex iPLEX Gold reaction cocktail (96 to 384 dispensing)” on page 84.

To prepare the high plex iPLEX Gold reaction cocktail (same DNA, different multiplexed assays for 96 to 384 dispensing)1. In a 1.5 mL tube, prepare the high plex iPLEX Gold reaction cocktail, without



Note: Primer mix (8 μM: 10 μM: 12 μM) will be added in the next step. 8 μM, 10 μM and 12 μM illustrate the three tiered concentration of the low to high mass primers. Low mass primers should be at 0.84 uM, medium mass primers should be 1.04 μM and high mass primers at 1.25 μM in the final 9 μL reaction.





5. Proceed to “To add the high plex iPLEX Gold reaction cocktail (96 to 384 dispensing)” on the next page.

Table 16: Multiplexed high plex iPLEX Gold reaction cocktail


Volume (768rxns)*









To add the high plex iPLEX Gold reaction cocktail (96 to 384 dispensing)1. Place the 96-well cocktail microtiter plate onto position 1 of the liquid-handler

deck.

2. Remove both 384-well sample microtiter plates from the thermal cycler. Centrifuge the cocktail microtiter plate at 1000 RPM for one minute. When complete, remove the plate sealing film.

3. Place the 384-well sample microtiter plates on plate flatteners. Then, place them on deck position 3 and 4.


5. When prompted for the number of plates, type 2 in the Edit Global Values dialog box.

2 μL of high plex iPLEX Gold reaction cocktail is added to each well of the sample microtiter plates.

1 2

3 4



1 2

3 4


Sample

Wash Station

Sample Sample


Cocktail




6. When the method is complete, remove and discard the 96-well microtiter plate of high plex iPLEX Gold reaction cocktail.

7. Proceed to “Thermocycling the High Plex iPLEX Gold Reaction in Two Plates” on the next page.

1 2

3 4


Sample

Wash Station

Sample Sample


Cocktail

Diagram of the liquid-handler deck configured for cocktail addition to 2 plates



Processing High iPLEX Gold Reactions for Two Destination Plates(19plex - 36+plex)Thermocycling the High Plex iPLEX Gold Reaction in Two Plates

Thermocyclingthe High Plex

iPLEX GoldReaction inTwo Plates

After you have added the high plex iPLEX Gold reaction cocktail to the 384-well sample microtiter plates (see the preceding section), the next step is to process the high plex iPLEX Gold reaction by thermocycling.

To thermocycle the high plex iPLEX Gold reaction1. Remove the 384-well sample microtiter plates and plate flatteners from position 3

and 4 on the liquid handler deck.



3. Centrifuge the 384-well sample microtiter plates at 1000 RPM for one minute.

4. Thermocycle the 384-well sample microtiter plates as follows:




3

5 cycles40 cycles



Processing High iPLEX Gold Reactions for Two Destination Plates(19plex - 36+plex)Thermocycling the High Plex iPLEX Gold Reaction in Two Plates

Note: If you have a programmable thermal cycler, enter the preceding program and name it iPLEX Gold Extend. When processing other iPLEX reactions in the future, simply run the iPLEX Gold Extend program for this step.

5. Proceed to “Cleaning Up the High Plex iPLEX Gold Reaction Products” on the next page..

Note: The cleanup of high plex iPLEX Gold reaction products involves adding water and then Clean Resin to the sample microtiter plate. If you are not ready to add Clean Resin, you may store the sample microtiter plates. Seal the microtiter plates with plate-sealing film and store it at -20º C.

Caution: When you are ready to add Clean Resin, let the microtiter plates thaw to room temperature before adding the resin.


Processing High iPLEX Gold Reactions for Two Destination Plates(19plex - 36+plex)Cleaning Up the High Plex iPLEX Gold Reaction Products

Cleaning Upthe High Plex


After the high plex iPLEX Gold reaction has been processed by thermocycling (see the preceding section), the next step is to clean up the high plex iPLEX Gold reaction products with Clean Resin (resin). This cleanup step is important to optimize mass spectrometry analysis of the high plex iPLEX Gold reaction products.


• Spread Clean Resin onto the 384-well dimple plate.• Add nanopure water to each well of the 384-well sample microtiter plates.• Add Clean Resin to the 384-well sample microtiter plates.• Rotate and centrifuge the 384-well sample microtiter plates.



Also, when not in use, keep the bottle of Clean Resin tightly closed. This is necessary to prevent the drying out of the resin.






4


384-Well dimple plate:384 wells arranged in a 16 X 24 grid

DImple PlateUse the 3mg dimple plate twice, or use a 6 mg dimple plate (Part Number 11235).



Resin

Scraper









deck.

2. When the 384-well microtiter plates are done thermocycling, centrifuge the 384-well sample microtiter plates at 1000 RPM for one minute. Remove them from the thermal cycler and remove the plate sealing film.


1 2

3 4





3. Place the 384-well sample microtiter plates on plate flatteners. Then, put them on position 3 and 4 of the liquid-handler deck.


5. When prompted for How Many 384 Plates, type 2 to indicate two destination plates have been placed on the liquid handler deck.

16 µL of water is added to the sample microtiter plates.

6. Remove the 384-well sample microtiter plates and flatteners from positions 3 and 4.

7. Seal the 384-well sample microtiter plates with plate sealing film. Then, remove the microtiter plates from their plate flattener.s


8. Centrifuge the microtiter plates (for 30 seconds at 1600 RPM, or until the air bubbles are gone).

9. Proceed to “To add resin to the sample microtiter plate” on the next page..

1 2

3 4


Sample

Wash Station

Sample Sample


Water Reservoir




To add resin to the sample microtiter plate

Note: Perform the following steps for each of the two sample microtiter plates.

1. Gently place the sample microtiter plate, upside-down, onto the dimple plate.



3. Repeat these steps for the second sample microtiter plate.

4. Once both sample microtiter plates have had resin added, proceed to “To rotate and centrifuge the high plex iPLEX Gold reaction products” below.


Dimple plate






Processing High iPLEX Gold Reactions for Two Destination Plates(19plex - 36+plex)Nanodispensing

To rotate and centrifuge the high plex iPLEX Gold reaction products1. Rotate the sample microtiter plates on a rotator for five minutes, at room

temperature.


2. Centrifuge the sample microtiter plates at 3200 g for five minutes.

3. The high plex iPLEX Gold reaction products are now ready for transfer to a SpectroCHIP, using the MassARRAY Nanodispenser.

Note: If you are not ready to transfer the high plex iPLEX Gold reaction products to a SpectroCHIP, you can store the microtiter plates at -20º C until you are ready. Place adhesive sealing foil (Marsh Biomedical Products, Inc. #AB-0626) on the microtiter plates before storing. Make sure the edges of the sealing foil are well-sealed. Do not store the microtiter plates for more than two weeks.

If you have stored microtiter plates of high plex iPLEX Gold reaction products, thaw, rotate, and then centrifuge the microtiter plates (at 3200 g for five minutes) before transferring the reaction products to a SpectroCHIP.

Nanodispensing Once liquid handling is complete, perform nanodispensing of high plex iPLEX Gold reaction products onto a SpectroCHIP. For instructions, see the MassARRAY Nanodispenser User’s Guide (Samsung).

DefiningAssays and

Plates


AcquiringSpectra



Appendix AExtension Primer Adjustment

Methods

Due to the inverse relationship between peak intensity and analyte mass, it is recommended that extend primers in iPLEX assays are adjusted by concentration to address this issue. The highest mass primer (~8500 Da) has a peak intensity 25% less than the average of the lower mass primers. Because of this, analyte signal-to-noise ratio estimation throughout the spectrum can pose a significant challenge to the Caller software. In the context of a genotyping reaction, analyte peaks can be missed, leading to genotyping errors.

In addition to the inverse relationship between analyte mass and signal-to-noise ratio, non-predictable variations in peak heights can occur. These variations may stem from inconsistent oligonucleotide quality and poor desorption/ionization behavior in MALDI. Optical density measurements to assess oligonucleotide quality are not sufficient since fragments from partial synthesis and other absorbing contaminants contribute to the over-estimation of full-length oligonucleotide concentration. Over-estimation of primer concentration prompts over-dilution, resulting in lower signal intensity in the spectrum and genotype calling problems.

To overcome this situation, testing primer mixes in MALDI-TOF MS prior to use for genotyping is recommended. A mixture of extend primers containing each primer at the same concentration is analyzed on the MassARRAY platform. TYPER Analyzer software can create a primer adjustment report that identifies primers to adjust within the mix and recommends a ratio for adjustment (see the “Primer Adjustment Report” section in Appendix A of iPLEX MassARRAY Typer User's Guide).

Given the inverse relationship between analyte mass and peak signal-to-noise ratio, the primer adjustment report specifies the high mass primers to be adjusted. To streamline the process flow, SEQUENOM proposes the following three options for preparing iPLEX primer cocktails:

• Option A: Doubling concentration of high and low mass primers in two groups as presented below:

Primer Adjustment Methods—Double 2-step Method (Option A)

Low Mass High Mass


• Option B: Doubling concentration of high and low mass primers in four groups, where the first group of low mass primers are mixed at 625 nM, the following group at 830 nM, the third group at 1.04 uM, and the higher mass group at 1.25 uM final reaction concentration 9 μl as shown below:

• Option C: Regression method, in which every primer is mixed at a specific concentration according to the mass (see the figure (B) on page 96).

Option A is the easiest method to use and is favored for a simpler reaction setup process; however, this may result in lower call rates. For maximal call rates, option B or C is preferred.

If option B is selected, the following procedure is recommended:

For 24 plexes, order oligonucleotides in four separate plates at the following concentrations:

• The first plate, containing the first six lower mass primers, at 168 uM each (7 uM x plex level),

• The second plate, containing the next six primers, at 223 uM each (9.3 uM x plex level)

• The third plate, containing the following six primers, at 278 uM each (11.6 uM x plex level)

• The fourth plate, containing the last six primers, at 336 uM each (14 x plex level)

The Plate Editor includes a dialog box that helps you determine these concentrations. For more information, see the “Extend Primer Adjustment” section in the “Plate Editor” chapter of the iPLEX MassARRAY Software User’s Guide.

Combine all four plates using the same volumes. Using 0.804 uL of this mixture in a 9μl iPLEX reaction would produce equilibrated spectra as shown in the figure above. At different plex levels, starting stock concentrations would be different. Multiply the values in parentheses by the plex level to obtain the desired oligonucleotide stock concentration before combining them into one plate.

If Option C is selected, the following procedure is recommended. Knowing the mass, it is possible to equilibrate each primer in the spectrum using specific concentrations. The following relation can be derived:

Intensity = -301Ln(Mass) + 2731

1st Group 2nd Group 3rd Group 4th Group

Primer Adjustment Methods—Double 4-step Method (Option B)


This relation does not consider oligonucleotide purity and non-predictable sequence-specific desorption/ionization variance; however, it is still useful in providing a good starting point. A Microsoft Excel spreadsheet can be created to calculate concentration and dilution to apply to each primer within a reaction. For example, apply the following formula to calculate the diluted oligo concentration (found in column F when exported to Excel from the Extention Primer Adjustment table found in the PlateEditor):

(LN(Mass)-7.82) * oligo stock concentration = target primer concentration in mixture

Knowing the initial concentration and volume, additional water volume (uL) can be calculated (column H). Then the equivalent volume of diluted primers can be used to create the working mix.

See the “Extension Primer Adjustment” section, found in the “Plate Editor” chapter within the iPLEX Software Guide for further instructions.

The figure (A) below shows a 23-plex equimolar iPLEX primer mix before adjustment. The last seven oligos, and particularly the one at ~7700 Da, have intensities close to detection limits. An iPLEX reaction on a heterozygous sample with below average yields would produce allelic peaks below detection thresholds. The figure (B) below shows the same primers mixed using the regression method (option C). This method, in combination with the primer adjustment report in TYPER, offers the best chance of success.

Inte

nsity

Mass (Da)

Relation of Peak Intensity and Mass


There are several ways to perform primer adjustment for iPLEX assays. SEQUENOM has proposed three methods, but there are other options. Selecting the appropriate method can help improve genotyping call rates and accuracy. This will improve study efficiency in the long run and therefor reduce costs.

A. Non-adjusted Equimolar Primer Mix

B. Adjusted with Regression Method

Comparison of Non-adjusted 23-plex Primer Mix Versus Adjusted Mix Using Option C, Regression Method


Appendix BTerms and Conditions

LIMITED LIABILITY

SEQUENOM shall not be liable, to any extent whatsoever, for any damages resulting from or arising out of the use or performance of this system and related documentation or the procedures specified in this manual, regardless of foreseeability or the form of action, whether in contract, tort (including negligence), breach of warranty, strict liability or otherwise, and including but not limited to, damages resulting from loss of data, loss of anticipated profits, or any special, indirect, incidental or consequential damages. In no event shall SEQUENOM's liability to the user exceed the amount paid by the user to SEQUENOM hereunder. The USER assumes full responsibility for the results obtained from the use of this system and related documentation and for application of such results. This system is for research use only and is not to be used for diagnostic purposes.

THE USER IS HEREBY PUT ON NOTICE THAT SEQUENOM’S MASSARRAY PRODUCTS AND iPLEX™ METHODS AND PROCESSES HAVE NOT BEEN SUBJECTED TO REGULATORY REVIEW OR APPROVED BY THE FEDERAL FOOD AND DRUG ADMINISTRATION OR ANY OTHER UNITED STATES GOVERNMENTAL AGENCY OR ENTITY, AND HAVE NOT BEEN APPROVED FOR CLIA COMPLIANCE, OR OTHERWISE APPROVED UNDER ANY STATUTE, RULE, LAW, OR REGULATION, FOR ANY PURPOSE, RESEARCH, COMMERCIAL, DIAGNOSTIC, OR OTHERWISE.

LIMITED WARRANTY

Limited Warranty Relating to MassARRAY Products. SEQUENOM warrants that the MassARRAY System will be free from defects in materials and workmanship and will conform to SEQUENOM’s current specifications, and perform accordingly, from the time of installation and for a period of one (1) year thereafter, so long as the MassARRAY System remains unchanged and in the original condition supplied by SEQUENOM. SEQUENOM warrants that the MassARRAY Kits will be free from defects in materials and workmanship and will conform to SEQUENOM’s specifications and perform accordingly up to the expiration date specified on the MassARRAY Kit packaging, so long as the MassARRAY Kits are stored according to specifications, and remain unchanged and in the original condition supplied by SEQUENOM. The foregoing warranty does not include periodic maintenance or calibration recommended for some MassARRAY Products. This warranty does not apply to defects resulting from improper or inadequate maintenance or calibration by the USER; defects resulting from hardware, software, interfacing, or supplies provided by parties other than SEQUENOM; defects resulting from unauthorized modification, maintenance, or repair, or improper use or operation outside of SEQUENOM’s specifications for the MassARRAY Products or by personnel not authorized by SEQUENOM, and; defects resulting from abuse, negligence, accident, loss or damage in transit. In addition, this warranty does not apply to damage due to (1) environmental conditions at the site of installation; (2) operator failure to perform standard operating procedures and routine maintenance as prescribed in the operator manuals; (3) moving (by other than SEQUENOM authorized personnel) the MassARRAY System from its installed location; (4) exposure of the MassARRAY Products to Bio-Safety Level 3 or 4 (as defined by the United States Occupational Health and Safety Administration) agents; or (5) exposure to radioactivity.


SEQUENOM’s sole obligation and liability for any breach of the limited warranty set forth herein shall be at SEQUENOM’s sole discretion and option: (1) to replace the MassARRAY Products, in whole or in part, provided that the USER notifies SEQUENOM of the defects, SEQUENOM directs the USER to return the defective MassARRAY Products to SEQUENOM and the USER returns the MassARRAY Products as directed, at SEQUENOM’s expense; or (2) to repair (and recalibrate as necessitated by repair) the MassARRAY Products in whole or in part. MassARRAY Products may not be returned to SEQUENOM under any circumstances without SEQUENOM's prior authorization. SEQUENOM shall not be liable, to any extent whatsoever, for any damages resulting from or arising out of the use or performance of the MassARRAY Products provided regardless of foreseeability or the form of the cause of action, whether in contract, breach of warranty, tort (including negligence, strict liability, or otherwise), and including but not limited to damages resulting from loss of data, loss of anticipated profits or revenue, or any special, direct, indirect, incidental or consequential damages.

The limited warranty sets forth SEQUENOM’s sole and exclusive responsibility with respect to any alleged breach of this limited warranty. Except as provided herein, the MassARRAY Products are provided without warranty of any kind or nature. SEQUENOM does not warrant, guarantee, or make any representations regarding the use or the results of the use, of the MassARRAY Products in terms of correctness, accuracy, reliability, or otherwise. The USER assumes the entire risk as to the results and performance of the MassARRAY Products. The foregoing warranty is exclusive and is made in lieu of and to the exclusion of any other warranties, whether oral or written, express or implied, direct, indirect, by estoppel or otherwise, or created by the Uniform Commercial Code or the usage in the industry or the course of dealings of the parties, as to any matter whatsoever, including but not limited to those concerning merchantability or fitness for a particular purpose.

LICENSES

Sequenom's patented DNA analysis by mass spectrometry methods (including the MassEXTEND® method) are protected under U.S. patent rights including but not limited to 6,500,621; 6,300,076; 6,258,538; and 5,869,242 and their foreign equivalent patent rights. With the purchase of Sequenom SpectroCHIP® array chips (whether purchased as a stand-alone product or in a kit product with reagents), Customer is granted a limited, one time use only per each chip element, right to practice MassEXTEND® methods. Such license right granted is limited to one-time use for the number of elements provided per SpectroCHIP® array chip purchased. For example, Sequenom's SpectroCHIP® 384 array chip is provided with 384 elements. Sequenom's SpectroCHIP® 96 array chip is provided with 96 elements. Such license right is granted for customer's internal research and development purposes only, and not for commercial use. Customer may not practice Sequenom's patented methods for providing services to third parties in exchange for fees or other consideration, without written permission from Sequenom. Re-manufacture or re-use of Sequenom's SpectroCHIP® array chips and elements, with Sequenom's patented DNA analysis methods, is prohibited. Other than as expressly set forth above, no license rights are granted or implied.

SOFTWARE LICENSES

For purposes of this section, "Software" means computer software or programs supplied by Sequenom to the Customer under this Agreement including but not limited to such

iPLEX™ Gold Application Guide 98 November 10, 2006 (Matrix, Samsung, Compact) Doc.11555 R00, CO 060190

software that is embedded in or forms an integral part of Sequenom's hardware products in addition to separately provided software for application specific or other purposes. For purposes of this section, Software does not include Oracle Corporation software products provided by Sequenom as such Oracle products are separately governed by the attached Oracle End User License Terms. (a) Sequenom grants Customer a non-exclusive and non-transferable license to use the Software for processing data for business purposes only. Customer shall not permit another party to use the Software and Customer shall effect and maintain adequate security measures to safeguard the Software from access or use by unauthorized persons. Customer shall not transfer, rent, lease, sublicense, loan, copy, modify, adapt, merge, translate, reverse engineer, decompile, or disassemble the Software or create derivative works based on the whole or any part of the Software. (b) The Software license shall not be deemed to extend to any of Sequenom's intellectual property rights, including rights in source code. No copies may be made of the Software without the prior written consent of Sequenom, except that Customer may make a single back up or archival copy. The Software license shall apply to any copy as it applies to the Software. (c) With specific reference to Sequenom's iPLEXTM application software (the "iPLEXTM Software") that is part of Sequenom's Typer 3.3 software product, Customer is provided free of charge, a royalty-free, non-exclusive and non-transferable license to use the iPLEXTM Software subject to the terms and conditions in subsections (a) and (b) above and elsewhere in this section, solely upon the condition that such iPLEXTM Software is used exclusively in conjunction with the specially designated reagent kit product (catalog item no. 10130) purchased from Sequenom. License rights to use the iPLEXTM Software are not granted by or with the iPLEXTM Software by itself, separate and apart from use in conjunction with such specially designated reagent kit product purchased from Sequenom. All Software licenses shall terminate automatically and immediately if Customer fails to abide by any of the terms in this section or under this Agreement.

CONFLICTS

In the event that a conflict arises between the language of this Proposal and any work order, purchase order, billing statement, or invoice related to the purchase, license, or other transfer of any SEQUENOM products or technology or services to be provided, the language of this Proposal shall govern and control, and the conflicting terms, provisions, and conditions of any such other documents shall be deemed non-existent, and shall not obligate or be binding upon Customer or SEQUENOM.


iPLEX™ Gold Application Guide 100 November 10, 2006 (Matrix, Samsung, Compact) Doc.11555 R00, CO 060190

Documents

iPLEX Gold Application Guide-111006 - University of Chicagocancer-seqbase.uchicago.edu/documents/iPLEXGold... · 2006. 11. 13. · Title: iPLEX Gold Application Guide-111006.book