Ovation WGA FFPE System - BMLabosisSPIA Buffer S01341 Red DB2 SPIA Enzyme S01342 Red DB3 Table 6. Additional Reagents COMPONENT 6200-08 PART NUMBER VIAL CAP VIAL NUMBER Nuclease-free

U S E R G U I D E

Ovation® WGA FFPE SystemPART NO. 6200-08

2 Ovation WGA FFPE System

Patents, Licensing and Trademarks

© 2010 NuGEN® Technologies, Inc. All rights reserved. The Ovation® and Applause™ families of products and methods are covered by U.S. Patent Nos. 6,692,918, 6,251,639, 6,946,251 and 7,354,717, and other issued and pending patents in the U.S. and other countries. NuGEN, the NuGEN logo, Ovation, SPIA, Ribo-SPIA, WT-Ovation, Applause, Encore, Prelude and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners. QIAGEN® and Sensiscript® are registered trademarks of QIAGEN GmbH, Hilden Germany, licensed to NuGEN. QIAGEN components contained herein are developed and manufactured by QIAGEN GmbH, Hilden Germany. Licensed under U.S. Patent No. 5,814,506. This product is sold under license from bioMérieux under US 5,824,517 and foreign counterparts.

The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right (without the right to modify, reverse engineer, resell, repackage or further sublicense) under these patent applications and any patents issuing from these patent applications to use this product and methods, accompanying this package insert, for research and development purposes solely in accordance with the intended use described and the written instructions provided in this user guide. No license to make or sell products by use of this product is granted to the buyer whether expressly, by implication, by estoppels or otherwise. In particular, the purchase of this product does not include or carry any right or license to use, develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including commercial services or clinical diagnostics.

For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research, please contact NuGEN Technologies, Inc., 201 Industrial Road, Suite 310, San Carlos, CA 94070. Phone (888-654-6544 or 650-590-3600); FAX (888-296-6544 or 650-590-3630).

Warranty

NuGEN warrants that this product meets the performance standards described in the Company’s product and technical literature for a period of six months from the date of purchase, provided that the product is handled and stored according to published instructions, and that the product is not altered or misused. If the product fails to meet these performance standards, NuGEN will replace the product free of charge or issue a credit for the purchase price.

NuGEN’s liability under this warranty shall not exceed the purchase price of the product. NuGEN shall assume no liability for direct, indirect, consequential or incidental damages arising from the use, results of use or inability to use its products. NuGEN reserves the right to change, alter or modify any product to enhance its performance and design.

NuGEN’s products are developed, designed and sold FOR RESEARCH USE ONLY. This product is not to be used for diagnostic or drug purposes, nor is it to be administered to humans or animals. Except as expressly set forth herein, no right to modify, reverse engineer, distribute, offer to sell or sell NuGEN’s product is conveyed or implied by buyer’s purchase of this NuGEN product. The buyer agrees to use NuGEN products accompanying the product insert in accordance with the intended use and the written instructions provided.


Table of Contents

Contents

I. Introduction ......................................................................................................... 1A. Background ....................................................................................................... 1B. WGA FFPE SPIA Technology ............................................................................ 1C. Performance Specifications ............................................................................... 2D. Quality Control ................................................................................................. 2E. Storage and Stability ......................................................................................... 2F. Material Safety Data Sheet (MSDS) ................................................................... 3

II. Components ........................................................................................................ 4A. Reagents Provided ............................................................................................ 4B. Additional Equipment, Reagents and Labware ................................................ 5

III. Planning the Experiment ..................................................................................... 7A. Input DNA Requirements .................................................................................. 7B. Using Nuclease-free Techniques ....................................................................... 8C. SPIA Product Storage ........................................................................................ 8

IV. Protocol ............................................................................................................... 9A. Overview ........................................................................................................... 9B. Protocol Notes .................................................................................................. 9C. Agencourt® RNAClean® XP Purification Beads ............................................... 10D. Programming the Thermal Cycler ................................................................... 12E. Fragmentation ................................................................................................. 13F. End Repair ....................................................................................................... 14G. A-tailing ........................................................................................................... 14H. Purification of A-tailed gDNA ......................................................................... 15I. Adaptor Ligation ............................................................................................. 16J. Purification of SPIA Template .......................................................................... 16K. SPIA Amplification .......................................................................................... 17L. Purification of SPIA Product ............................................................................ 18M. Measuring SPIA Product Yield and Purity ....................................................... 19

V. Technical Support .............................................................................................. 20

VI. Appendix ........................................................................................................... 21A. Purification Protocols for SPIA Product ........................................................... 21B. Labeling and Hybridization to Agilent CGH Arrays ........................................ 23C. Quality Control of Amplified SPIA Product ..................................................... 24D. Preventing Non-specific Amplification ............................................................ 25E. Library Preparation for Next Generation Sequencing (NGS) .......................... 26F. Frequently Asked Questions (FAQs) ............................................................... 27


A. Background

The Ovation® WGA FFPE System provides a fast and simple method for preparing lin-early amplified DNA (SPIA Product) from genomic DNA prepared from Formalin Fixed Paraffin Embedded (FFPE) tissue. The resulting amplified DNA may be used in a variety of downstream applications including, array CGH (aCGH) applications and sequencing library construction (requires the Encore™ ds-DNA Module; Part No. 2500-08). Because of the simplicity and robustness of the procedure, accurate and quantitative results are obtained with minimal dropouts and excellent coverage.

The Ovation WGA FFPE System is powered by SPIA® technology, a rapid, simple and robust nucleic acid amplification process developed by NuGEN. Starting with 100 ng of genomic DNA from FFPE tissue, microgram quantities of amplified SPIA product can be prepared in approximately six hours.

The Ovation WGA FFPE System (Part No. 6200-08) provides optimized reagent mixes to process eight FFPE DNA samples for downstream labeling and hybridization on aCGH arrays, or construction of libraries for next generation sequencing using the Encore ds-DNA Module (Part No. 2500-08). It is recommended to include an appropri-ate control DNA samples when first using this product (not provided).

B. WGA FFPE SPIA Technology

WGA FFPE ligation-mediated SPIA is a 5-step process that generates amplified DNA (SPIA product) from 100 ng of FFPE gDNA.

1. Fragmentation (35 minutes)FFPE gDNA is enzymatically fragmented to reduce the overall size and enable full coverage by the amplification process.

2. End Repair (1 hour)Fragmented dsDNA is repaired to produce uniform blunt ends.

3. A-tailing (1.25 hours)Adenine residues are added to the 3´ ends of the end-repaired dsDNA to facilitate Adaptor ligation.

4. Adaptor Ligation (1.25 hours)Adaptors containing target hybridization sequences for Single Primer Isothermal Amplification (SPIA) are ligated to the end-repaired, A-tailed dsDNA to create SPIA templates.

I. Introduction


I. Introduction

5. SPIA (1.75 hours)SPIA® is a linear isothermal DNA amplification process developed by NuGEN. It uses a DNA/RNA chimeric SPIA primer, DNA polymerase and RNase H in a homogeneous isothermal assay that provides highly efficient amplification of DNA sequences. SPIA primer hybridizes to the target sequence present on the SPIA Adaptors ligated previously onto each end of the double-stranded SPIA template. DNA polymerase then initiates replication at the 3´ end of each primer, displacing the existing strands. The RNA portion at the 5´ end of the newly synthesized strand is again removed by RNase H, exposing part of the unique priming site for initiation of the next round of synthesis. The process of SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage is repeated, resulting in rapid accumulation of DNA with a sequence complementary to the original template.

C. Performance Specifications

The Ovation WGA FFPE System synthesizes microgram quantities of amplified SPIA product starting with total FFPE DNA input amounts of 100 ng. In approximately six hours, the Ovation WGA FFPE System can produce 3.5 to 5 µg of amplified DNA ready for labeling and analysis by aCGH or conversion to double-stranded DNA and sub-sequent sequencing library construction using the Encore ds-DNA Module (Part No. 2500-08) and Encore NGS Library System I (Part No. 300-08), Encore NGS Multiplex System I (Part No. 301-32) or other suitable library construction methods compatible with input of double-stranded DNA.

D. Quality Control

Each Ovation WGA FFPE System lot is tested to meet specifications of amplification performance.

E. Storage and Stability

The Ovation WGA FFPE System is shipped on dry ice and should be unpacked immedi-ately upon receipt.

Note: This product contains components with multiple storage temperatures.

All kit components, except the Beckman Coulter Genomics’ Agencourt® RNAClean® XP Beads (clear cap), should be stored at –20°C in a freezer without a defrost cycle.

The vial labeled Agencourt RNAClean XP Beads (clear cap) should be removed from the top of the shipping carton upon delivery and stored at 4°C.

The Ovation WGA FFPE System has been tested to perform to specifications after as many as six freeze/thaw cycles. Kits handled and stored according to the above guide-lines will perform to specifications for at least six months. NuGEN has not yet established long-term storage conditions for the Ovation WGA FFPE System.


I. Introduction

F. Material Safety Data Sheet (MSDS)

An MSDS for this product is available from NuGEN Technical Service by calling 888-654-6544 or by sending an email to: [email protected].


A. Reagents Provided

Table 1. Fragmentation Reagents

COMPONENT6200-08

PART NUMBERVIAL CAP VIAL NUMBER

Fragmentation Buffer S01343 Violet F1 ver 2

Fragmentation Enzyme S01344 Violet F2 ver 2

Table 2. End Repair Reagents

COMPONENT6200-08


End Repair Buffer S01345 Blue ER1 ver 2

End Repair Enzyme S01346 Blue ER2 ver 2

Table 3. A-tailing Reagents

COMPONENT6200-08


A-tailing Buffer S01347 Blue AT1

A-Tailing Enzyme S01348 Blue AT2

Table 4. Ligation Reagents

COMPONENT6200-08


Ligation Buffer S01337 Yellow L1 ver 2

Ligation Adaptor S01338 Yellow L2 ver 2

Ligation Enzyme S01339 Yellow L3 ver 3

II. Components


II. Components

Table 5. SPIA Reagents

COMPONENT6200-08


SPIA Primer S01340 Red DB1

SPIA Buffer S01341 Red DB2

SPIA Enzyme S01342 Red DB3

Table 6. Additional Reagents

COMPONENT6200-08


Nuclease-free Water S01001 Green D1

Agencourt® RNAClean® XP Beads

S01307 Clear —

B. Additional Equipment, Reagents and Labware

Required Materials

• Equipment - Microcentrifuge for individual 1.5 mL and 0.5 mL tubes - 0.5–10 µL pipette, 2–20 µL pipette, 20–200 µL pipette, 200–1000 µL pipette - Vortexer - Thermal cycler with 0.2 mL tube heat block, heated lid, and 100 µL

reaction capacity - Appropriate spectrophotometer and cuvettes, or Nanodrop® UV-Vis

Spectrophotometer• Reagents

- Ethanol (Sigma-Aldrich, Cat. #E7023), for purification steps• Supplies and Labware

- Nuclease-free pipette tips - 1.5 mL and 0.5 mL RNase-free microcentrifuge tubes - 1.5 mL amber, DNase-free microcentrifuge tubes - 0.2 mL individual thin-wall PCR tubes or 8 X 0.2 mL strip PCR tubes or

0.2 mL thin-wall PCR plates


II. Components

- Beckman Coulter Genomics SPRIPlate® 96R, Ring Magnet Plate (Beckman Coulter Genomics, Cat. #A29164) or Beckman Coulter Genomics SPRIPlate Ring Super Magnet Plate, (Beckman Coulter Genomics, Cat. #A32782).

- MagnaBot® II Magnetic Separation Device (Promega, Cat. #V8351). Other magnetic separation systems may be used as well, although their perfor-mance has not been validated by NuGEN.

- Purification options for final SPIA Product purification (select one option): ° Agencourt RNACleanXP Kit (Beckman Coulter Genomics, Cat. #A63987) ° QIAquick® PCR Purification Kit (QIAGEN, Cat. #28104) ° QIAGEN MinElute

- Disposable gloves - Kimwipes - Ice bucket

• Optional Materials - Agilent 2100 Bioanalyzer or materials and equipment for electrophoretic

analysis of nucleic acid - Real-time PCR system - Cleaning solutions such as RNaseZap® (Ambion, Cat. #AM9780) and

DNA-OFF™ (MP Biomedicals, Cat. #QD0500)

To Order:

• Ambion Inc., www.ambion.com• Beckman Coulter Genomics, www.beckmangenomics.com• Invitrogen Life Technologies, www.invitrogen.com• MP Biomedicals, www.mpbio.com• New England BioLabs, www.neb.com• QIAGEN Inc., www.qiagen.com• Sigma-Aldrich, Inc., www.sigmaaldrich.com• USB Corporation, www.usbweb.com


A. Input DNA Requirements

1. DNA QuantityDNA input is 100–200 ng. Lower amounts of input will potentially result in insufficient yields or insufficient genomic coverage depending on required analytical platforms.

2. DNA PurityDNA samples must be free of contaminating proteins and other cellular material, organic solvents (including phenol and ethanol) and salts used in many DNA isola-tion methods. Use of a commercially available system for preparing small amounts of DNA that does not require organic solvents is recommended.

If a method including organic solvents is used, we recommend column purifica-tion after isolation. Methods that use RNases to degrade RNA co-isolated with the desired DNA can be problematic as the presence of RNase in the gDNA sample can inhibit amplification, resulting in low yields and/or decreased detection of copy number changes.

One measure of DNA purity is the ratio of absorbance readings at 260 and 280 nm. The A260:A280 ratio for DNA samples should be in excess of 1.8. DNA samples with lower ratios may result in low amplification yield.

3. DNA IntegrityThe Ovation WGA FFPE System was designed and optimized for use with highly degraded DNA samples typically isolated from FFPE blocks The ligation-mediated SPIA approach allows low-quality, highly degraded DNA samples to be ampli-fied successfully. We strongly recommend quantitation of total DNA to assure the minimum input requirement is met. We recommend analysis of FFPE DNA samples on either an Agilent Bioanalyzer or an appropriate gel electrophoresis system to determine the level of degradation. FFPE DNA samples that are much less degraded and whose size distribution is considerably higher than those shown in Figure 1 will not perform well without additional fragmentation.

III. Planning the Experiment


III. Planning the Experiment

Figure 1. Five representative FFPE gDNA samples with optimal size distribution for use in the Ovation WGA FFPE System. The DNA molecular weight standard shown in the first lane is the 50 bp ladder from Invitrogen run on a 1.2% agarose gel with sizes (nt) indicated.

2652 >

800 >

350 >

4. Carrier Use for DNA IsolationWe strongly recommend against the use of nucleic acid based carriers during DNA purification because many have been shown to interfere with SPIA template synthesis. For the latest information regarding other carriers, contact our technical services team.

B. Using Nuclease-free Techniques

Nuclease contamination from equipment and the work environment will lead to experimental failure. Follow these guidelines to minimize contamination:

• Wear disposable gloves and change them frequently.• Avoid touching surfaces or materials that could introduce nucleases.• Use only the reagents provided and recommended.• Clean work areas and instruments, including pipettes, with commercially avail-

able decontamination reagents.• Use only new nuclease-free pipette tips and microcentrifuge tubes.

C. SPIA Product Storage

The amplified SPIA Product produced by the Ovation WGA FFPE System may be stored at –20°C for at least 6 months.


A. Overview

The WGA SPIA amplification process used in the Ovation WGA FFPE System is performed in five stages:

1. gDNA fragmentation: 0.5 hour

2. End repair: 1 hour

3. A-tailing: 1.25 hours

4. SPIA adaptor ligation: 1.25 hours

5. SPIA isothermal linear amplification and purification: 1.75 hours

Total time to prepare amplified SPIA product ~6 hours

Ovation WGA FFPE System components are color-coded, with each color linked to a specific stage of the process. Performing each stage other than ligation requires mak-ing a master mix, then adding it to the reaction, followed by incubation. Master mixes are prepared by mixing components provided for that stage.

B. Protocol Notes

• We recommend the routine use of a good quality positive control DNA. Especially the first time you set up an amplification reaction, the use of a posi-tive control DNA will allow the establishment of a baseline of performance and provide the opportunity to become familiar with the bead purification step. This step may be unfamiliar to many users and can be especially prone to handling variability in using the magnet plate, so a practice run with the plate is highly recommended. Set up no fewer than 4 reactions at a time with the 6200-08 kit. This ensures sufficient reagent recoveries for 8 total amplifications from a single kit. Making master mixes for fewer than 4 samples at a time may affect reagent recovery volumes.

• Due to the high sensitivity inherent in this amplification system, it is required that users take measures to minimize the potential for the carryover of previ-ously amplified SPIA Product into new amplification reactions. The two steps to accomplish this are: 1. Designate separate workspaces for “pre-amplification” and “post-amplification” steps and materials, and 2. Implement routine clean-up protocols for workspaces as standard operating procedure. Detailed instruc-tions are listed in the Appendix.

• Thaw components used in each step and immediately place them on ice. It is best not to thaw all reagents at once.

• Use the water provided with the kit (green: D1) or an alternate source of nuclease-free water. We do not recommend the use of DEPC-treated water with this protocol.

• Always keep thawed reagents and reaction tubes on ice unless otherwise instructed.

• After thawing and mixing buffer mixes, if any precipitate is observed, re-dissolve the precipitate completely prior to use. You may gently warm the buffer mix for

IV. Protocol


IV. Protocol

2 minutes at room temperature followed by brief vortexing. Do not warm any enzyme or primer mixes.

• When placing small amounts of reagents into the reaction mix, pipet up and down several times to ensure complete transfer.

• When instructed to pipet mix, gently aspirate and dispense a volume that is at least half of the total volume of the reaction mix.

• Always allow the thermal cycler to reach the initial incubation temperature prior to placing the tubes or plates in the block.

• When preparing master mixes, use the minimal amount of extra material to ensure 8 reactions in the kit. The Ovation WGA FFPE System Quick Protocol will automatically calculate an appropriate overfill volume which can be used as a guideline in setting up master mixes.

• Components and reagents from other Ovation System products should not be used with this product.

• Use only fresh ethanol stocks to make ethanol for washes in the DNA purifica-tion protocols. Make the ethanol mixes fresh as well. Lower concentrations of ethanol in wash solutions will result in loss of yield, as the higher aqueous con-tent will dissolve the DNA and wash it off the beads or column.

C. Agencourt® RNAClean® XP Purification Beads

Tips and Notes:

There are significant modifications to the Beckman Coulter Genomics Agencourt® RNAClean® XP beads’ standard procedure; therefore, you must follow the procedures outlined in this User Guide for the use of these beads with the Ovation WGA FFPE System. However, you may review the Beckman Coulter Genomics user guide to become familiar with the manufacturer’s recommendations.

The bead purification processes used in this kit consist of the following steps:

1. Binding of DNA to RNAClean XP beads

2. Magnetic separation of beads from supernatant

3. Ethanol wash of bound beads to remove contaminants

4. Elution


IV. Protocol

Figure 2. Bead purification process overview.

Additional Tips and Notes

• Remove beads from 4°C and leave at room temperature for at least 15 minutes. Before use, ensure that they have completely reached room temperature. Cold beads will result in reduced recovery.

• Fully resuspend beads by inverting and tapping before adding to the sample.• Note that we recommend using different volumes of RNAClean XP beads than

the standard Beckman Coulter Genomics protocol. • It is critical to let the beads separate on the magnet for the full amount of time

listed in the protocol. Removing the binding buffer before the beads have com-pletely separated will impact yields.

• After the binding step has been completed, it is important to minimize bead loss when removing the binding buffer. With the samples placed on the magnet, remove only the amount specified in the protocol of the binding buffer from each sample. Some liquid will remain at the bottom of the tube, but this will minimize bead loss.

• Any significant loss of beads bound to the magnet during the ethanol washes will impact yields, so make sure the beads are not lost with the wash.

• Ensure that the 70% ethanol wash is freshly prepared from fresh ethanol stocks. Lower percent ethanol mixes will reduce recovery.

• During the ethanol washes, keep the samples on the magnet. The beads should not be allowed to disperse; the magnet will keep the beads on the walls of the sample wells or tubes in a small ring.

• It is critical that all residual ethanol be removed prior to continuing with the protocol. Therefore, when removing the final ethanol wash, first remove most of the ethanol, then allow the excess to collect at the bottom of the tube before removing the remaining ethanol. This reduces the required bead air-drying time.

• After drying the beads for the recommended amount of time, inspect each tube carefully and make certain that all the ethanol has evaporated before proceeding.

• It is strongly recommended that strip tubes or partial plates are firmly placed when used with the magnetic plate. We don’t advise the use of individual tubes, as they are not very stably supported on the magnetic plates.


IV. Protocol

D. Programming the Thermal Cycler

Use a thermal cycler with a heat block designed for 0.2 mL tubes, equipped with a heated lid, and with a capacity of 100 µL reaction volume. Prepare the programs shown in Table 7, following the operating instructions provided by the manufacturer. For ther-mal cyclers with an adjustable heated lid, set the lid temperature at 100°C. For thermal cyclers with a fixed-temperature heated lid (e.g., ABI GeneAmp® PCR 9600 and 9700 models) use the default settings (typically 100 to 105°C).

Table 7. Thermal Cycler Programming

FRAGMENTATION

Program 1Fragmentation

4°C – 1 min, 37°C – 15 min, 65°C – 15 min, hold at 4°C

END REPAIR

Program 2 End Repair


A-TAILING

Program 3 A-tailing


SPIA ADAPTOR LIGATION

Program 4 SPIA Adaptor Ligation


SPIA

Program 5 SPIA Amplification



IV. Protocol

E. Fragmentation

Important Note: Carry out steps E (Fragmentation) through J (Purification of SPIA Template ) in a pre-amplification workspace using dedicated pre-amplification consumables and equipment. Wipe all surfaces, equipment and instrumentation with a DNA decontaminant solution such as DNA-OFF to avoid the potential introduction of previously amplified DNA into new amplifications. For more information on our requirements for workflow compartmentalization and routine lab cleanup please refer to Appendix D of this User Guide. If you have any questions on this important topic, please contact NuGEN Technical Services ([email protected], or 888-654-6544).

1. Obtain Fragmentation Buffer (violet: F1), Fragmentation Enzyme (violet: F2) and the water (green: D1) from the components stored at –20°C.

2. Spin down contents of F2 and place on ice.

3. Thaw the other reagents at room temperature. Mix by vortexing. Spin and place on ice. Place D1 at room temperature.

4. For each sample add 4 µL FFPE gDNA (100 ng) to a 0.2 mL PCR tube.

5. Prepare a master mix by combining F1 and F2 in a 0.5 mL capped tube, according to the volumes shown in Table 8.

Table 8. Fragmentation Master Mix (volumes listed are for a single reaction)

FRAGMENTATION BUFFER(VIOLET: F1 ver 2)

FRAGMENTATION ENZYME(VIOLET: F2 ver 2)

5.0 µL 1.0 µL

6. Add 6 µL of the Fragmentation Master Mix to each tube.

7. Mix by pipetting 6–8 times and spin.

8. Place tubes in a pre-cooled thermal cycler programmed to run Program 1 (Fragmentation; see Table 7):


9. Remove tubes from the thermal cycler, spin to collect condensation and place on ice.

10. Continue immediately with the End Repair protocol.

Mix by pipetting and spin down the master mix briefly. Place on ice. Use immediately.


IV. Protocol

F. End Repair

1. Obtain the End Repair Buffer (blue: ER1) and End Repair Enzyme (blue: ER2) from the components stored at –20°C.

2. Spin down contents of ER2 and place on ice.

3. Thaw reagent ER1 at room temperature, mix by vortexing, spin and place on ice.

4. Make a master mix by combining ER1 and ER2 in a 0.5 mL capped tube, according to the volumes shown in Table 9.

Table 9. End Repair Master Mix (volumes listed are for a single reaction)

END REPAIR BUFFER(BLUE: ER1 ver 2)

END REPAIR ENZYME (BLUE: ER2 ver 2)

9.0 µL 1.0 µL

5. Add 10 µL of the End Repair Master Mix to each Fragmentation reaction tube.

6. Mix by pipetting 6–8 times, spin and place on ice.

7. Place tubes in a pre-cooled thermal cycler programmed to run Program 2 (End Repair; see Table 7):



9. Continue immediately with the A-tailing protocol.

G. A-tailing

1. Remove the RNAClean XP beads from 4°C storage and place at room temperature.

2. Obtain the A-tailing Buffer (blue: AT1) and End Repair Enzyme (blue: AT2) from the components stored at –20°C.

3. Spin down contents of AT2 and place on ice.

4. Thaw reagent AT1 at room temperature, mix by vortexing, spin and place on ice.

5. Make a master mix by combining AT1 and AT2 in a 0.5 mL capped tube, according to the volumes shown in Table 10.


The purification beads should be removed from 4°C and left at bench top to reach room temperature well before the start of purification.


IV. Protocol

Table 10. A-tailing Master Mix (volumes listed are for a single reaction)

A-TAILING BUFFER(BLUE: AT1)

A-TAILING ENZYME (BLUE: AT2)

9.0 µL 1.0 µL

6. Add 10 µL of the A-Tailing Master Mix to each End Repair reaction tube.

7. Mix by pipetting 6–8 times, spin and place on ice.

8. Place tubes in a pre-cooled thermal cycler programmed to run Program 3 (A-tailing; see Table 7):



10. Continue immediately with the Purification of A-tailed gDNA protocol.

H. Purification of A-tailed gDNA

1. Thaw Adaptor Ligation Reagents (L, yellow cap vials). Place L1 ver 2 Ligation Buffer at room temperature. Place remaining Ligation Reagents on ice.

2. Prepare a fresh 70% ethanol solution. Make fresh ethanol solution each day from recently opened 100% ethanol stock. Prepare enough volume for this and subse-quent bead purification following Adaptor ligation.

3. Ensure the RNAClean XP beads have completely reached room temperature before proceeding.

4. Resuspend beads by inverting and tapping the tube. Ensure beads are fully resuspended before adding to sample. After resuspending, do not spin the beads. A large excess of beads is provided; therefore, it is not necessary to recover any trapped in the cap.

5. At room temperature, add 54 µL bead suspension to each reaction and mix by pipetting up and down 10 times. Incubate at room temperature for 15 minutes.

6. Transfer tubes to magnet and let stand 5 minutes to completely clear the solution of beads.

7. Carefully remove only 74 µL of the binding buffer and discard it. Leaving some of the volume behind minimizes bead loss at this step.

Note: The beads should not disperse; instead, they will stay on the walls of the wells as a small ring. Significant loss of beads at this stage will impact yields, so ensure beads are not removed with the binding buffer or the wash.

8. With the samples still on the magnet, add 200 µL of freshly prepared 70% ethanol and allow to stand for 30 seconds.


Minimize bead loss by leaving a residual volume of binding buffer after comple-tion of the binding step.

Best results can be obtained by using fresh 70% ethanol in the wash step.


IV. Protocol

9. Remove the 70% ethanol wash using a pipette.

10. Repeat the 70% ethanol wash 2 more times, for a total of 3 washes.

Note: With the final wash, it is critical to remove as much of the ethanol as pos-sible. Use at least 2 pipetting steps to allow excess ethanol to collect at the bot-tom of the tubes before removing the last of the ethanol.

11. Air-dry the beads on the magnet for 10 minutes. Inspect each tube carefully to ensure that all the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing with elution.

12. Remove samples from magnet. Add 23 µL L1 Ligation Buffer, pipette up and down to fully resuspend beads.

13. Incubate at room temperature for 3 minutes.

14. Transfer samples to magnet, let stand for 3 minutes.

15. Remove 22 µL of the eluate from each A-tailing reaction tube while still on the magnet and place in fresh reaction tubes.

16. Continue immediately with the Adaptor Ligation protocol.

I. Adaptor Ligation

1. Add 2 µL L2 ver 2 Adaptor to each reaction tube. Mix by pipetting 6–8 times.

2. Add 1 µL L3 ver 3 Ligation Enzyme to each reaction tube, mix by pipetting 6–8 times, spin and place on ice.

3. Place tubes in a thermal cycler programmed to run Program 4 (SPIA Adaptor Ligation; see Table 7):


4. Remove samples from thermal cycler, spin and place tubes on bench top.

5. Continue immediately with the Purification of SPIA Template protocol.

J. Purification of SPIA Template

1. Ensure the RNAClean XP beads have completely reached room temperature before proceeding.

2. Resuspend the RNAClean XP beads by inverting and tapping the tube. Ensure beads are fully resuspended before adding to sample. After resuspending, do not spin the beads. A large excess of beads is provided; therefore, it is not necessary to recover any trapped in the cap.

3. At room temperature, add 40 µL bead suspension to each reaction and mix by pipetting up and down 10 times. Incubate at room temperature for 10 minutes.

The purification beads should be removed from 4°C and left at bench top to reach room temperature well before the start of purification.


IV. Protocol

4. Transfer samples to magnet and let stand 5 minutes to completely clear the solu-tion of beads.

5. Carefully remove only 55 µL of the binding buffer and discard it. Leaving some of the volume behind minimizes bead loss at this step.

Note: The beads should not disperse; instead, they will stay on the walls of the wells as a small ring. Significant loss of beads at this stage will impact yields, so ensure beads are not removed with the binding buffer or the wash.

6. With the samples still on the magnet, add 200 µL of freshly prepared 70% ethanol and allow to stand for 30 seconds. Prepare the 70% ethanol fresh on the day of your experiment.

7. Remove the 70% ethanol wash using a pipette.

8. Repeat the 70% ethanol wash 2 more times, for a total of 3 washes.

Note: With the final wash, it is critical to remove as much of the ethanol as pos-sible. Use at least 2 pipetting steps to allow excess ethanol to collect at the bot-tom of the tubes before removing the last of the ethanol.

9. Air-dry the samples on the magnet for 10 minutes. Inspect each tube carefully to ensure that all the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing with the SPIA Amplification protocol.

K. SPIA Amplification

1. Obtain the SPIA Primer Mix (red: DB1), SPIA Buffer Mix (red: DB2) and SPIA Enzyme Mix (red: DB3) stored at –20°C.

2. Thaw reagent DB1 and DB2 at room temperature, mix by vortexing, spin and place on ice.

3. Thaw DB3 on ice and mix the contents by inverting gently 5 times. Ensure the enzyme is well mixed without introducing bubbles, spin and place on ice.

4. Make a master mix by sequentially combining DB2, DB1 and DB3 in a 0.5 mL capped tube according to the volumes shown in Table 11. Make sure the addition of DB3 is at the last moment.

Table 11. SPIA Master Mix (volumes listed are for a single reaction)

SPIA BUFFER(RED: DB2)

SPIA PRIMER (RED: DB1)

SPIA ENZYME (RED: DB3)

20 µL 10 µL 10 µL

5. Add 40 µL of the SPIA master mix to each tube containing the SPIA Template bound to the dried beads. Use a pipette set to 30 µL and mix well by pipetting up

Minimize bead loss by leaving a residual volume of binding buffer after comple-tion of the binding step.

Best results can be obtained by using fresh 70% ethanol in the wash step.



IV. Protocol

and down at least 8–10 times. Attempt to get the majority of the beads in suspen-sion and remove most of the beads from the tube walls.

Note: Beads may not form a perfectly uniform suspension, but this will not affect the reaction. The addition of SPIA master mix will elute the template off the beads.

6. Place tubes in a pre-cooled thermal cycler programmed to run Program 5 (SPIA Amplification; see Table 7):



Important Note: At this point the tubes or plate must be removed from the pre-amplification workspace. Carry out all remaining steps in a post-amplification workspace using dedicated post-amplification consumables and equipment. Take care to avoid the introduction of previously amplified SPIA Product into your pre-amplification workspace. For more information on our requirements for workflow compartmentalization and routine lab cleanup, please refer to Appendix D of this User Guide. If you have any questions on this important topic, please contact NuGEN Technical Services ([email protected], (888) 654-6544).

8. Transfer samples to magnet and let stand 5 minutes to completely clear the solu-tion of beads.

9. Carefully transfer the cleared supernatant containing the amplified SPIA Product to fresh tubes. The beads may now be discarded.

10. Proceed immediately with the Purification of SPIA Product protocol or store reac-tion products at –20°C overnight prior to continuing.

L. Purification of SPIA Product

Amplified SPIA product can be purified using various methods listed in Appendix A. Purification is required if the amplified SPIA Product is intended for use in subsequent labeling reactions or for input into the Encore ds-DNA Module workflow.

Selection of the optimum purification method can depend on many factors. Please contact the NuGEN Technical Support team for assistance in selecting the appropriate purification option for your application.


IV. Protocol

M. Measuring SPIA Product Yield and Purity

1. Mix the sample by brief vortexing and spinning prior to checking the concentration.

2. Measure the A260, A280 and A320 of your amplified SPIA product. You may need to make a 1:20 dilution of the DNA in water prior to measuring the absorbance.

3. Purity: Subtract the A320 value from both A260 and A280 values. The adjusted A260–A320/A280–A320 ratio should be >1.8.

4. Yield: Assume 1 A260 of ssDNA = 33 µg/mL. To calculate:(A260–A320 of diluted sample) X (dilution factor) X 33 (concentration in µg/mL of 1 Abs solution) X 0.03 (final volume in mL) = total yield in micrograms

5. Alternatively, you may measure the concentration and purity of SPIA DNA with a Nanodrop, using 1 absorbance unit at 260 nm of ssDNA = 33 µg/mL as the constant.

6. The purified SPIA product may be stored at –20°C, labeled for subsequent aCGH analysis, or made double-stranded and used for library construction.


For Technical Support, please contact NuGEN at (U.S. only) 888.654.6544 (Toll-Free Phone) or 888.296.6544 (Toll-Free Fax) or email [email protected].

In Europe contact NuGEN at +31(0)135780215 (Phone) or +31(0)135780216(Fax) or email at [email protected].

In all other locations, contact your NuGEN distributors Technical Support team.

V. Technical Support


A. Purification Protocols for SPIA Product

There are three currently supported alternatives for carrying out the final purification of SPIA Product. Listed alphabetically, they are: 1) Beckman Coulter Genomics’ Agencourt RNAClean XP Kit, 2) the QIAGEN MinElute Reaction Cleanup Kit, and 3) the QIAGEN QIAquick® PCR Purification Kit.

The procedures given below are specifically adapted for use with NuGEN products and may differ significantly from the protocols published by the manufacturers. Failure to fol-low the purification procedures as given below may negatively impact your results.

Beckman Coulter Genomics’ Agencourt RNAClean XP Kit, Cat. #A63987 (instructions for a single reaction)

1. Obtain and shake the RNAClean XP bottle to resuspend the magnetic beads.

2. Transfer the 40 µL SPIA Product into a clean 1.5 mL tube, 0.2 mL strip tubes or PCR plate.

3. Add 72 µL of resuspended RNAClean XP beads to the 40 µL DNA sample.

4. Mix the sample and beads thoroughly by pipetting up and down 10 times.

5. Incubate samples at room temperature for 5 minutes.

6. Place the tubes or plate on the plate magnet for 10 minutes or until solution clears.

7. Using a multi-channel pipette, remove and discard the supernatant. Do not disturb the ring of magnetic beads.

8. With the reaction plate still on the plate magnet, add 200 µL of freshly prepared 80% ethanol to each well of the reaction plate and incubate for 30 seconds or until the solution clears. Add slowly as to not disturb the separated magnetic beads.

9. Using a multi-channel pipette, remove and discard the ethanol.

10. Repeat the 80% ethanol wash 2 more times. Ensure all the ethanol is removed from the plate.

11. Air-dry the tubes or plate at bench top away from the plate magnet for no more than 2 minutes. If the beads dry too long, they become difficult to resuspend.

12. With the reaction vessel at bench top, add 30 µL nuclease-free water (green: D1) to each tube or well. Vortex for 30 seconds. Ensure the beads are fully resuspended. Vortex longer if necessary.

13. Replace reaction vessel back on the plate magnet. Allow the beads to separate for 5 minutes or until the solution clears.

14. Using a pipette, remove the eluted sample and place into a fresh tube.

Note: Small amounts of magnetic bead carryover may interfere with sample quan-titation. Take care to minimize bead carryover.

VI. Appendix


VI. Appendix

15. Collect sample. There should be approximately 30 µL of purified SPIA Product.

16. Mix sample by vortexing, spin briefly.

17. Proceed to Measuring SPIA Product Yield and Purity protocol or store DNA at –20°C.

QIAGEN MinElute Spin Column, Cat. #28204 (instructions for a single reaction)

1. Into a clean 1.5 mL tube, add 300 µL of ERC buffer from the QIAGEN kit.

2. Add the entire amount of amplified SPIA Product product to the tube. Mix sample by vortexing, then spin briefly.

3. Obtain one MinElute spin column and insert into a collection tube.

4. Load the entire solution of sample and buffer onto column.

5. Centrifuge for 1 minute at maximum speed. Discard flow-through.

6. Place column back in the same collection tube. Add 750 µL of Buffer PE with ethanol.

7. Centrifuge for 1 minute at maximum speed. Discard flow-through.

8. Centrifuge again for 1 minute at maximum speed to remove all residual Buffer PE with ethanol.

9. Remove the MinElute spin column from the centrifuge. Discard flow-through along with the collection tube.

10. Blot the column tip onto a filter paper to remove any residual wash buffer from the tip of the column.

Note: Blotting of the column tip MUST be done prior to transferring the column to a clean 1.5 mL microcentrifuge tube. Failure to do so may result in a small quantity of wash buffer in your final eluted sample.

11. Place the MinElute spin column in clean 1.5 mL collection tube.

12. Add 30 µL of nuclease-free water to the center of each MinElute spin column. Do not use cold water!

13. Let columns stand for 1 minute at room temperature.

14. Centrifuge at maximum speed for 1 minute.

15. Collect sample. There should be approximately 30 µL of purified SPIA Product.

16. Mix sample by vortexing, then spin briefly.


Use nuclease-free water at room temperature to elute sample.


VI. Appendix

QIAGEN QIAquick PCR Purification Kit, Cat. #28104 (instructions for a single reaction)

1. Into a clean 1.5 mL tube add 200 µL of PB buffer from the Qiagen system.

2. Add the 40 µL of amplified SPIA product to the tube.


4. Obtain 1 QIAquick spin column and insert into a collection tube.

5. Load 240 µL of sample onto the column.

6. Centrifuge column in a collection tube for 1 minute at 13,000 rpm (~17,900 X g).

7. Discard flow-through. Place the column back in the same collection tube.

8. Add 700 µL of 80% ethanol.

Note: Use fresh 80% ethanol. Lower percent ethanol mixes will reduce recovery.

9. Centrifuge the column for 1 minute at 13,000 rpm (~17,900 X g). Discard flow-through.

10. Repeat steps 8 and 9 once.

11. To remove remaining liquid, centrifuge column for 1 additional minute at 13,000 rpm (~17,900 X g).

12. Remove the column from the centrifuge. Discard flow-through with the collection tube.

13. Blot the tip of the column onto a filter paper in order to remove any residual wash buf-fer from the tip of the column.

Note: Blot column tip prior to transferring it to a new tube to prevent any wash buffer transferring to the eluted sample.

14. Place the column in a clean 2.0 mL collection tube, appropriately labeled.

15. Add 30 µL of nuclease-free water (green: D1) to the center of each column.

16. Let columns stand for 5 minutes at room temperature to elute purified SPIA Product.

17. Centrifuge at 13,000 rpm (~17,900 X g) for 1 minute to collect sample. There should be approximately 30 µL of purified SPIA Product.



B. Labeling and Hybridization to Agilent CGH Arrays

NuGEN recommends labeling 1.5–2.0 µg of SPIA product with the Invitrogen BioPrime® Total for Agilent® aCGH system (Invitrogen Cat. #A10963-010 or A10963-011) or BioPrime® Total Genomic Labeling System (Invitrogen Cat. #18097-010 or 18097-011) according to manufacturer’s instructions. SPIA product does not need to


VI. Appendix

be restriction digested prior to labeling. Users of Agilent 4x44k and 8x15k CGH Arrays may elute the labeled SPIA product in a smaller volume of 30 µL.

Follow instructions in Agilent CGH manual G4410-90010 for array set up, hybridization and wash.

C. Quality Control of Amplified SPIA Product

The size distribution of the amplified SPIA product can be a useful tool for assess-ing the quality of the material with an Agilent Bioanalyzer. Note that the shape of this distribution trace can be dependent on the input DNA integrity as well as DNA source. We recommend using a DNA 1000 LabChip (Agilent Cat. #5067-1504 ) following the manufacturer’s instructions. A typical size distribution trace may look like the one depicted in Figure 3 below.

Figure 3. Bioanalyzer trace of Amplified SPIA product obtained from 100 ng input of FFPE breast tumor (red trace) and FFPE liver (green trace) gDNA

FFPE Breast Tumor FFPE Liver


VI. Appendix

D. Preventing Non-specific Amplification

Due to the high sensitivity inherent in our amplification systems, we have devel-oped a set of recommendations designed to minimize the potential for the gen-eration of non-specific amplification products by carryover of previously amplified SPIA DNA. We strongly recommend implementing these procedures, especially for high-throughput environments typical in today’s laboratories. We have two general recommendations. First, designate separate workspaces for “pre-amplification” and “post-amplification” steps and materials. This provides the best work environment for processing FFPE DNA using our highly sensitive amplification protocols. Our sec-ond recommendation is to implement routine clean-up protocols for workspaces as standard operating procedure. This will prevent amplification products from spread-ing through laboratory workspaces. Details regarding establishing and maintaining a suitable work environment are listed below:

1. Designate a pre-amplification workspace separate from the post-amplification workspace or general lab areas:

a. Pre-amplification includes all steps and materials related to FFPE DNA sample handling and dilution, NuGEN’s Fragmentation, End-repair, A-tailing, A-tailed DNA Purification, Ligation, SPIA Template Purification and SPIA Amplification. After SPIA incubation the reactions are immediately removed from the pre-amplification workspace and opened only in the post-amplification area.

b. Post-amplification includes all steps and materials related to the handling of the final amplified SPIA product, including bead removal, final purification, double-stranded DNA generation, array hybridization, library construction, and any other analytical work.

c. Ideally, the pre-amplification workspace will be in a separate workroom. If this is not possible, ensure the pre-amplification area is sufficiently isolated from post-amplification work.

d. We recommend the use of “PCR Workstation” enclosures with UV illumination as pre-amplification workspaces where conditions preclude physical separation of pre- and post-amplification activities.

2. Establish and maintain a clean work environment:

a. Initially clean the entire lab thoroughly with DNA-OFF.

b. In the pre-amplification area, remove all small equipment, and then clean every surface that may have been exposed to amplified SPIA Product (surfaces, drawer handles, keypads, etc.). Before reintroducing any equipment, clean every piece of equipment thoroughly. It is important to clean wells of thermal cyclers and mag-netic plates with a cotton swab or by filling with cleaning solution.

c. Always wear gloves, and don fresh gloves upon entry into this controlled area. Frequently change gloves while working in the pre-amplification area, especially prior to handling stock reagents, reactions and FFPE DNA samples.


VI. Appendix

d. Stock this area with clean (preferably new) equipment (pipettes, racks, consum-ables) that has not been exposed to post-amplification workspace.

e. Make it a policy to carry out regular cleaning of all workspaces.

f. Capture waste generated in both pre- and post-amplification areas (tips, columns, wash solutions from beads and columns, tubes, everything) in sealable plastic bags and dispose of them promptly after each experiment to avoid waste spillage.

g. Do not open amplified product reaction vessels in the pre-amplification workspace.

h. Avoid running negative controls.

E. Library Preparation for Next Generation Sequencing (NGS)

NuGEN’s Encore ds-DNA Module is required to make double-stranded DNA used in NGS library construction. For users of the Illumina sequencing platforms, material produced by the Encore ds-DNA Module may be used as input to the Encore NGS Library Systems to prepare DNA libraries for sequencing. For use on Illumina plat-forms, we offer the Encore NGS Library System I (Part No. 300-08) for non-multiplex sequencing, and Encore NGS Multiplex System I (Part No. 301-32) to enable multi-plexing of up to eight samples per lane.

Alternatively, reagents for performing the Library Construction steps for Illumina sequencing may be obtained from other vendors. The double-stranded SPIA Product produced by the Ovation WGA FFPE System and Encore ds-DNA Module may also be used with other leading NGS platform providers such as Life Technologies and Roche 454. Please contact these companies for recommendations on suitable library construction methods.


VI. Appendix

F. Frequently Asked Questions (FAQs)

Q1. What materials are provided with the Ovation WGA FFPE System? The Ovation WGA FFPE System provides all necessary primers, buffers and enzymes for Fragmentation, End Repair, A-tailing, Adaptor Ligation and SPIA amplification.

Q2. What equipment is required or will be useful? The Beckman Coulter Genomics’ SPRIPlate 96R Ring Magnet Plate, SPRIPlate Ring Super Magnet Plate, or MagnaBot® II Magnetic Separation Device (Promega, Cat. #V8351) is required for the template purification step. Other required equipment includes a microcentrifuge, pipettes, vortexer, thermal cycler and a UV/Vis spectrophotometer. An Agilent Bioanalyzer or a similar instrument may be used for quality control.

Q3. What additional consumables does the user need? For the SPIA Product purification step, purification columns or magnetic beads are required.

Q4. What is the polymerase and fidelity of the assay? The DNA polymerase used in the SPIA amplification reaction is a standard strand-displacement polymerase. Due to the linear nature of the amplifi-cation technology, SPIA has inherently higher fidelity compared to other non-linear procedures. This is because each amplified product is generated from an original template, thereby, amplification products themselves are not amplifiable. As a result, the process prevents reproduction of the promulga-tion of an error introduced in earlier cycles, a source of concern in exponen-tial amplification systems.

Q5. What DNA can be used as a positive control for the amplification system?We routinely use 20 ng of intact gDNA (Promega Cat. #G4171).

Q6. What is the length of the amplified SPIA Product? The amplified SPIA Product from good quality DNA ranges from 50 nt to 400 nt, with an average size of approximately 200 nt. and can vary depend-ing upon the sample type.

Q7. How long does the assay take to complete? Approximately six hours.

Q8. Does the Ovation WGA FFPE System amplify GC-rich genomic regions? We have not identified any regions of genomic DNA that the kit systemati-cally fails to amplify.

Q9. How can I label SPIA products for CGH analysis? NuGEN has obtained high-quality data using the Invitrogen BioPrime DNA labeling system for use on Agilent’s CGH arrays. We have also used the Agilent Genomic DNA Enzymatic Labeling Kit (Cat. #5190-0449).

VI. Appendix

NuGEN Technologies, Inc.

Headquarters USA

201 Industrial Road, Suite 310 San Carlos, CA 94070 USA Toll Free Tel: 888.654.6544 Toll Free Fax: 888.296.6544 [email protected]@nugeninc.com

Europe

P.O. Box 1496680 AC Bemmel The Netherlands Tel: +31-13-5780215Fax: [email protected]

For our international distributors contact information, visit our website

www.nugeninc.com

Q10. How much SPIA product should be used for labeling for aCGH?NuGEN typically uses 2 µg of SPIA product for labeling with the use of the 4x44k array format.

Q11. For aCGH , what is the best choice of reference DNA that I should amplify when designing my experiment?NuGEN has seen the best aCGH results with the reference DNA that matches the experimental DNA as closely as possible. Ideally this sample is obtained from the native adjacent tissue (NAT) and preserved using the same fixation protocol and storage conditions as the experimental sample. If a matched NAT reference is not available, we recommend obtaining a similarly aged, gender mis-matched, non-tumor FFPE DNA from a commercial source such as BioChain (Hayward, CA) or Asterand (Detroit, MI).

Q12. Can the Ovation WGA FFPE System be used for SNP genotyping? Yes. Sequencing error rates are very similar in samples processed with Ovation WGA FFPE System and Encore ds-DNA Module relative to unampli-fied material, indicating there is no increase in sequencing errors contributed by the amplification process that could impact SNP calls.

Q13. What genomic DNA isolation methods are compatible with the Ovation WGA FFPE System? Various genomic DNA isolation techniques that produce high-quality and high-purity gDNA should be compatible with NuGEN Ovation WGA FFPE System. QIAGEN QIAamp DNA FFPE Tissue Kit (Cat. #56404) was used extensively in the development of the Ovation WGA FFPE System. Follow manufacturer’s recommendations carefully to obtain optimum results.

Q14. What are the recommended storage conditions for the SPIA Product? SPIA Product may be stored at –20°C. Ensure the vials are well sealed and avoid multiple freeze-thaw cycles.

Q15. Are labeling reagents included in the Ovation WGA FFPE System? No. This kit only includes the reagents necessary for generating SPIA Product (amplified DNA). Labeling and hybridization reagents must be purchased separately.

M01174 v1

©2010 NuGEN Technologies, Inc. All rights reserved. The Ovation® and Applause™ families of products and methods are covered by U.S. Patent Nos. 6,692,918, 6,251,639, 6,946,251 and 7,354,717, and other issued and pending patents in the U.S. and other countries. NuGEN, the NuGEN logo, Ovation, SPIA, Ribo-SPIA, WT-Ovation, Encore, Applause, Prelude and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners.

For research use only.

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Ovation WGA FFPE System - BMLabosisSPIA Buffer S01341 Red DB2 SPIA Enzyme S01342 Red DB3 Table 6. Additional Reagents COMPONENT 6200-08 PART NUMBER VIAL CAP VIAL NUMBER Nuclease-free