Integrated Solutions. for Nucleic. Acid. Purification

Transcription

1 Integrated Solutions for Nucleic Acid Purification

2 Table of Contents Genomic DNA 3 Plasmid DNA 15 Fragment DNA 25 Total RNA 35 You have a choice for Nucleic Acid Purification The yield and concentration you need Application-tested protocols Mini Mega scale Manual or Automated format Global support and service DNA and RNA Purification Systems from scientists for scientists, for all your perfomance and throughput needs. 2

3 Purify High-Quality DNA......from a broad range of sample types with easy-to-use, labor saving systems. Promega genomic DNA purification systems provide: Increased productivity: Save time and labor with our easy to use protocols. Yield: Our systems and methods provide high DNA yields and purity. Performance: Sample-type specific protocol proven for PCR/genotyping. Choice of throughput: Select the format (manual single-tube, manual plate or automated plate/tube) to meet your requirements. Convenience: Choose a kit that processes multiple sample types or for challenging samples, a specific sample-type kit. Sample types: Blood Bacteria Mouse tail and animal tissue Tissue culture cells Plants Food Applications for other sample types Genomic DNA How can we help you achieve your goals today? A Comparison of Genomic DNA Purification Systems. Mouse Tails Tissue Plant and Animal Culture Tissue or Manual Automated Spin Vacuum Magnetic Blood Bacteria Tissue Cells Food Handling Handling Protocol Protocol Technology Wizard Genomic DNA Purification System (Cat.# A1120, A1125, A1620) Wizard SV Genomic DNA Purification System (Cat.# A2360, A2361) Wizard SV 96 Genomic DNA Purification System (Cat.# A2370, A2371) MagneSil Blood Genomic, Max Yield System (Cat.# MD1360) MagneSil ONE, Fixed Yield Blood Genomic System (Cat.# MD1370) Wizard Magnetic 96 DNA Plant System (Cat.# FF3760, FF3761) MagneSil Genomic, Fixed Tissue System (Cat.# MD1490) MagneSil KF, Genomic System (Cat.# MD1460) Wizard Magnetic DNA Purification System for Food (Cat.# FF3750, FF3751) 3

4 Wizard Genomic DNA Purification Kit High molecular weight gdna from multiple sample types in a scalable format. The Wizard Genomic DNA Purification Kit provides: Confidence: Proven salting out method - long track record. Performance: High-molecular weight DNA suitable for amplification, restriction enzyme digestion, and southern blotting from a wide range of starting materials. Scalability: Convenient for any sample size. Value: Cost per prep based on your sample size. Safe and quick: Non-organic solvent method can be completed in about 1 hour. DNA Yields from Various Starting Materials. Amount of Typical Source Starting Material DNA Yield Whole Blood 300µl 5 15µg 3ml 25 50µg 10ml µg 96-well plate, 50µl/well µg Tissue Culture Cells cells 5 30µg Animal Tissue Mouse Liver 11mg 15 20µg Mouse Tail 0.5 1cm of tail 10 30µg Insect Cells cells 16µg Plant Leaf Tissue 40mg 7 12µg Bacterial Culture* cells 5 20µg Yeast* cells µg *Overnight culture. Wizard Genomic DNA Purification Kit 100 isolations x 300µl A isolations x 300µl A isolations x 10ml A1620 For Laboratory Use. bp 10,000 3,000 1, µl 100µl 50µl 20µl M PCR analysis of genomic DNA isolated from decreasing amounts of whole blood. PCR products from 5µl of genomic DNA isolated using the Wizard Genomic DNA Purification Kit as the starting template. Lane M, 1kb DNA Ladder (Cat.# G5711); lane 1, no DNA control ( ); lane 2, Human Genomic DNA (Cat.# G3041) positive control (+). Lanes 3, 5, 7 and 9 contain samples that were rehydrated at room temperature for 20 minutes. Lanes 4, 6, 8 and 10 contain samples rehydrated at 4 C overnight. Sample Add Cell Lysis Solution. Incubate. Centrifuge. Discard supernatant. Vortex pellet. Add Nuclei Lysis Solution. Mix. Add Protein Precipitation Solution. 2862TB02_0A Centrifuge. Transfer supernatant to new tube containing isopropanol. Centrifuge. Discard supernatant. Add ethanol. Centrifuge. Aspirate ethanol. Air-dry pellet. Rehydrate DNA. Flow chart of genomic DNA purification using the Wizard Genomic DNA Purification Kit. 4

5 Wizard SV Genomic DNA Purification System Easy-to-use gdna isolation from cells or tissue in 20 minutes. The Wizard SV Genomic DNA Purification System provides: Speed: PCR-ready genomic DNA in as little as 20 minutes after lysis. Flexibility: Purify genomic DNA from 20mg of tissue, 1.2cm mouse tail or 5 x 10 6 cultured cells in a single prep. Yield: Purify 20 30µg of gdna per prep from 1.2cm mouse tail clips with an A 260 /A 280 ratio of 1.7. Wizard SV Genomic DNA Purification System 50 preps A preps A2361 Genomic DNA Purification from Mouse Tail Clipping or Tissue Sample Mouse tail clipping or tissue sample Proteinase K (Cat.# V3021)* digestion in Digestion Solution. Genomic DNA Purification from Tissue Culture Cells Wash tissue culture cells with 1X PBS. Genomic DNA M + Tail Liver Spin Heart Brain Tail Liver Vacuum Heart Brain Incubate at 55 C overnight (16 18 hours). Add Wizard SV Lysis Buffer. Transfer lystate to minicolumn. Add Wizard SV Lysis Buffer. Centrifuge. 3710TB04_2A Vacuum Adapter (Cat.# A1331)* Bind DNA. Amplification of genomic DNA isolated from various tissue sources using the Wizard SV Genomic DNA Purification System. One microliter of purified genomic DNA from the indicated tissues was amplified using PCR Master Mix (a) (Cat.# M7502) and mouse-specific IL-1ß primers (1.2kb product). Reactions with Mouse Genomic DNA (Cat.# G3091; +) and without DNA ( ) were performed as positive and negative controls, respectively. Vac-Man Laboratory Vacuum Manifold (Cat.# A7231)* Wash, removing solution by centrifugation or vacuum. Transfer spin column to a 1.5ml microcentrifuge tube (not provided). Centrifuge. Elute genomic DNA. 3643MA02_2A Flowchart of genomic DNA purification using the Wizard SV Genomic DNA Purification System. *May be purchased separately. (a) The PCR process is covered by patents issued and applicable in certain countries*. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. *In the U.S., effective March 29, 2005, U.S. Pat. Nos. 4,683,195, 4,965,188 and 4,683,202 will expire. In Europe, effective March 28, 2006, European Pat. Nos. 201,184 and 200,362 will expire. 5

6 Wizard SV 96 Genomic DNA Purification System Easy-to-use rapid 96-well isolation of gdna from cells or tissue. The Wizard SV 96 Genomic DNA Purification System provides: Performance: Purified DNA PCR ready. Time savings: 96-well processing using either manual or automated purification protocols for Beckman Coulter Biomek instruments Flexibility: Purify genomic DNA from up to 20mg of tissue, 1.2cm of mouse tail or cultured cells per well. Convenient: Purified genomic DNA from 96 mouse tails in minutes, and from cultured cells in 30 minutes after lysis. No centrifugation step required. PCR-ready gdna can be isolated from tissue or mouse tails directly from lysate. Wizard SV 96 Genomic DNA Purification System 1 x 96 preps A x 96 preps A2371 Average Yield of Genomic DNA Purified from Various Sources Using the Wizard SV and SV 96 Genomic DNA Purification Systems. Amount of Average Source Starting Material DNA Yield Mouse Tail Clipping 20mg 20µg Mouse Liver 20mg 15µg Mouse Heart 20mg 10µg Mouse Brain 20mg 6µg CHO Cells cells 5µg NIH3T3 Cells cells 9µg 293 Cells cells 8µg A B C D E F G H Add sample lysate. Bind DNA. Mouse Tail Clippings Water Only M C +C C4 C5 C6 C7 C8 D4 D5 D6 D7 D8 M E4 E5 E6 E7 E8 Wash. 3712TA04_2A No cross-contamination. Genomic DNA purified from mouse tail clipping samples or water samples were purified in adjacent wells of a 96-well plate. PCR products were amplified from 1µl of purified sample from each well using primers for mouse IL-1β. Ten microliters of PCR product were run on a 1.5% agarose gel and visualized by staining with ethidium bromide. Expected PCR product from mouse tail clippings for IL-1β is approximately 1.2kb. No PCR product was detected from water samples. Elute genomic DNA. Highly pure genomic DNA. 3637MA02_2A Flowchart of 96-well genomic DNA purification using the Wizard SV 96 Genomic DNA Purification System. 6

7 MagneSil Blood Genomic, Max Yield System Walk-away, high-throughput, automated gdna isolation from small volumes of blood in 1.5 hours! The MagneSil Blood Genomic, Max Yield System provides: Efficiency: Save labor using our walk-away high-throughput 96-well purification method. Consistent results: Maximum yield of 4 6µg of purified highquality genomic DNA from well-to-well and plate-to-plate. Time savings: Standardized protocol provides reproducible results in 1.5 hours. Let us program your robot downloadable methods available at: MagneSil Blood Genomic, Max Yield System 1 x 96 preps MD1360 For Laboratory Use. M bp 318 Genomic DNA Cleanup Tips WS1 Blood Sample Resin Disp XX XX XX Heat Block Heat Block PCR analysis of genomic DNA isolated from 200µl whole blood samples. PCR products from 5µl of genomic DNA isolated using the MagneSil Blood Genomic, Max Yield System. Lane M, PCR markers (Cat.# G3161); (+) positive control apc; ( ) no DNA control. Lanes 1 9 contain PCR amplification product from purified human genomic DNA. P10 Orbital1 P7 2nd Tips Lysis Resin MagnaBot Lysis Wash Lysis Disp Elution Solution XX XX XX XX XX XX P1 P4 P7 P11 P14 P18 3rd Tips Wash Plate Salt Wash Salt Disp Elution Disp 4 C 21 C 20 C M XX SWAP XX XX XX XX P2 XX XX XX XX P16 P20 P3 P5 P6 P8 P9 Deck layout for the MagneSil Blood Genomic Max Yield System on the Beckman Coulter Biomek FX. P12 4th Tips Elution Tips Alcohol Wash Alcohol Disp P13 P15 P MA05_3A Purification of apoptotic DNA using the MagneSil Blood Genomic, Max Yield System. In some cases, blood samples that are stored or shipped at elevated temperatures contain white blood cells undergoing apoptosis. The MagneSil Blood Genomic System purifies DNA across the broad size range seen in a typical apoptotic ladder. Genomic DNA was isolated from blood that had been stored at the indicated temperatures for 8 days. 4235TA07_3A 7

8 MagneSil ONE, Fixed Yield Blood Genomic System Walk-away, high-throughput fixed-yield genomic DNA isolation from small volumes of blood in 1 hour! The MagneSil ONE, Fixed Yield Blood Genomic System provides: Labor savings: Normalized fixed yield of 1µg eliminates the need for DNA quantitation, reducing the number of steps needed for genotyping. Consistent results: Obtain 1µg (fixed yield) of highly pure DNA from 60µl of anti-coagulated blood. Time savings: Complete walk-away high-throughput 96-well takes 1-hour. Place the blood samples onto the instrument and come back in an hour to purified and normalized DNA ready for PCR! MagneSil ONE, Fixed Yield Blood Genomic System 1 96 preps MD1370 For Laboratory Use. ng control Consistent yield of genomic DNA purified with the MagneSil ONE, Fixed Yield System. Genomic DNA standards were loaded onto the gel at the following concentrations: 250, 125, 63 and 31ng. Forty-eight blood samples were purified with the MagneSil ONE System, and 10µl of the 200µl total elution volume for each sample was loaded on the gel and visualized by ethidium bromide staining. 4231TA07_3A Cleanup Tips Heat Block SWAP P5 P9 P13 Heat XX Block P1 Wash Tips Wash Solution Alcohol Disp Alcohol Disp P17 WS1 SC1 XX XX XX XX XX P2 Working Plate P6 Lysis Wash P10 Lysis Disp P14 Elution Tips TR1 XX XX XX XX Magbead P3 P7 P11 P15 Resin Disp MagnaBot Lysis Resin Elution Solution Elution Disp XX XX XX XX XX Shaker1 Orbital1 P4 P8 P12 P MA05_3A Deck layout for the MagneSil ONE Fixed Yield Blood Genomic System on the Beckman Coulter Biomek FX. 8

9 Wizard Magnetic 96 DNA Plant System Easy, labor-saving 96-well isolation of high-quality plant gdna for PCR-based genotyping Wizard Magnetic 96 DNA Plant System provides: Time and labor savings: High-throughput manual or automated 96-well protocols give freedom from the time and labor-intensive steps normally associated with plant genomic DNA preps. Manual protocol takes about 45 minutes while the Beckman Coulter Biomek FX robot will complete purification in about 25 minutes. Consistent DNA quality: From well-to-well, plate-to-plate across a broad variety of plant material. Ease of use: Fewer steps and non-clogging magnetic particle technology. Adaptable chemistry: Scalable purification for standard or deep well plates. Modified chemistry for fixed yield purification and direct genotyping of gdna. Time Required to Isolate gdna from Plant Tissue Using Three Listed Manual Procedures. Wizard Magnetic 96 DNA Plant System 2 x 96 preps FF x 96 preps FF3761 Place fresh leaf or seed sample in 96 deep-well plate in the presence of Lysis Buffer A. Add 1 or 2 grinding beads. Grind. Centrifuge. Transfer supernatant to a clean plate. Add Lysis Buffer B/MagneSil Paramagnetic Particle mixture and mix well. Incubate. Place the plate on the MagnaBot 96 Magnetic Separation Device. Discard liquid. Genomic DNA Method Steps Time Wizard Magnetic 96 DNA Plant System minutes Commercial membrane plate minutes CTAB homebrew method minutes Examples of Plant Sample Types Successfully Processed Using the Wizard Magnetic 96 DNA Plant System. Arabidopsis Cotton seed Soybean Cabbage seed Grass seed Squash Canola leaf Green pepper seed Squash seed Canola seed Lettuce Strawberry leaf* Carrot seed Milkweed leaf Sunflower seed Chicory leaf Potato tuber Tobacco seedling Chives Radish leaf Tomato leaf Corn leaf Rice leaf Tomato seed Cotton leaf* Sorghum Watermelon seed * These samples need the addition of PVPP to the lysis buffer to remove phenolic compounds that inhibit PCR. Add Wash Buffer and mix. Place the plate on the MagnaBot 96 Magnetic Separation Device. Discard liquid. Repeat the wash. Dry the particles. Add Nuclease-Free Water. Place the plate on the MagnaBot 96 Magnetic Separation Device. Remove purified DNA to a fresh plate. 3220M A01_1A Flowchart of plant genomic DNA purification using the Wizard Magnetic 96 DNA Plant System. 9

10 MagneSil Genomic, Fixed Tissue System Easy-to-use isolation of high-quality DNA from fixedtissue and paraffin-embedded samples The MagneSil Genomic, Fixed Tissue System provides: Ease of use: No deparaffinization step with organic solvents. A manual method to purify DNA from thin sections of formalin-fixed, paraffin-embedded samples. High-quality DNA: Demonstrated amplification of fragments as large as 1,800bp. Quick results: Proteinase K treated sample processed to purified DNA in under 30 minutes, 6 12 samples in under 1 hour. Consistent amplification: Ultraclean DNA free of inhibitors of amplification including very small fragments of DNA. MagneSil Genomic, Fixed Tissue System 100 samples MD1490 For Laboratory Use. Add Incubation Buffer/ Proteinase K solution to tissue sample. Incubate overnight at 56 C. Add Lysis Buffer and Resin. Vortex. Place on Magnetic Stand and discard solution. A. Wash with Lysis Buffer. Wash with prepared 1X Wash Buffer. Repeat twice for a total of three washes. B. 926 base Size Marker 972 base Amelogenin Fragment Air dry Resin. C. Size Markers APC 1.8Kb Fragment 3931TB12_2A 65 C Add Elution Buffer. Heat. Analysis of DNA purified from paraffin-embedded, formalin-fixed 10µm thin sections using the MagneSil Genomic, Fixed Tissue System. Purified DNA was amplified and then analyzed on an ABI PRISM 3100 Genetic Analyzer. Panel A. Amplification with a set of 16 fluorescently labeled primers. Amplification products range in size from 104 to 420 bases. Panel B. A 972-base fragment amplified using an amelogenin primer set. Panel C. A 1.8kb fragment amplified from the APC gene. Results will vary depending on the degree of cross-linking due to formalin fixation. Remove tubes from heat, vortex, and place in Magnetic Stand. Transfer DNA to container of choice. 3742MA06_2A Flowchart of DNA isolation from formalin-fixed paraffin embedded tissue using the MagneSil Genomic, Fixed Tissue System. 10

11 MagneSil KF, Genomic System Walk away, low- to moderate-throughput, automated genomic DNA isolation in 25 minutes The MagneSil KF, Genomic System provides: Labor savings: Walk-away automation of 1 15 sample batch genomic DNA purification frees-up time for important work and cuts labor costs. Consistent results: Well-to-well 2 6µg of high-quality DNA per prep from 200µl of anti-coagulated whole blood. Simple method: One instrument and one chemistry for gdna purification, no training or modification of protocols necessary. Save time: Automated purification method takes 25 minutes to complete. MagneSil KF, Genomic System 200 preps MD1460 For Laboratory Use Yield gel of genomic DNA isolated from 200µl of liquid blood. Fifteen representative samples (single instrument run) of DNA purified using the MagneSil KF Genomic System from blood. Ten microliters of the 200µl total elution volume were loaded on the gel and visualized by ethidium bromide staining. Lanes 6 and 12 left intentionally blank. 4212TA06_3A Genomic DNA 4213TA06_3A Multiplex amplification of Factor II (345bp) and Factor V (225bp). Genomic DNA purified from 200µl human whole blood as recommended in Promega Technical Bulletin #TB322 MagneSil KF, Genomic System. Figure courtesy Dr. Greg Tsongalis, Hartford Hospital, USA. 11

12 Wizard Magnetic DNA Purification System for Food Labor-saving isolation of high-quality gdna from Food Wizard Magnetic DNA Purification System for Food provides: Ease of use: Protocol eliminates organic extraction. Time savings: No long incubations or centrifugation steps. Reliability: Quality controlled for PCR-based testing for GMO. Flexibility: Scaleable manual method to purify DNA from 200mg to 5g food samples. Consistent results: Ultraclean gdna of a broad size range free of inhibitors of amplification. Wizard Magnetic DNA Purification System for Food 200 preps FF preps FF3751 Prepare Lysate. Centrifuge. Bind DNA. Food Sample GMO Maize (%) bp M ,000 1,000 Capture MagneSil PMPs Duplicate Fluka maize reference standard maize samples containing up to 2% GMO maize were processed with the Wizard Magnetic DNA Purification System for Food. Ten percent of 100µl eluates of DNA in duplicate were loaded on a 1% agarose gel. The 0% standard shows a greater yield and higher molecular weight DNA because non-gmo maize undergoes minimal processing in the preparation of the reference standard. Lanes 1 and 2, 1kb DNA Step Ladder (Cat.# G6941) and PCR Markers (Cat.# G3161), respectively. 3019TA06_3A Wash MagneSil PMPs. Dry particles. Add sterile water. Vortex and incubate. bp 1, M 35S NOS C 35S Cornmeal (GMO+) NOS C 35S NOS C 35S Corn Seed (GMO ) NOS C 195bp (S35) 180bp (NOS) Capture particles. M DNA isolated from cornmeal and ground corn seed using the Wizard Magnetic DNA Purification System for Food. Detection of GMO sequences by PCR was performed using 1µl of DNA sample per template. The first three replicate sample sets (lanes 1 9) are from GMO+ cornmeal. Samples represented in lanes are from GMO corn seed; nonspecific bands are seen in these 35S and NOS lanes. Lane M, PCR Markers (Cat.# G3161). Lanes labeled C: chloroplast PCR positive controls. 3016TA06_3A Elute DNA. Flowchart of genomic DNA purification from food using the Wizard Magnetic DNA Purification System for Food. 3013MA07_0A 12

13 Ordering Information Wizard Genomic DNA Purification Kit* 100 isolations x 300µl A isolations x 300µl A isolations x 10ml A1620 Cell Lysis Solution* 1 liter A7933 Nuclei Lysis Solution* 50ml A liter A7943 Protein Precipitation Solution* 25ml A ml A7953 DNA Rehydration Solution* 50ml A7963 RNase A Solution, 4mg/ml* 1ml A7973 Proteinase K* 100mg V3021 Wizard SV Genomic DNA Purification System (a) 50 preps A preps A2361 Wizard SV 96 Genomic DNA Purification System (a) 1 x 96 preps A x 96 preps A2371 Proteinase K* 100mg V3021 Wizard SV Lysis Buffer* 50ml Z3052 Wizard SV Wash Solution* 185ml A1311 Neclei Lysis Solution* 50ml A7941 EDTA, 0.5M (ph 8.0), Molecular Grade* 100ml V4231 RNase A Solution, 4mg/ml* 1ml A7973 MagneSil Blood Genomic, Max Yield System (b) * 1 x 96 preps MD1360 Anti-Foam Reagent* 300µl MD1431 MagneSil Paramagnetic Particles* 25ml MD1441 Salt Wash, Blood* 90ml MD1401 Alcohol Wash, Blood* 70ml MD1411 Elution Buffer, Blood* 45ml MD1421 MagneSil ONE, Fixed Yield Blood Genomic System (b) * 1 x 96 preps MD1370 Lysis Buffer, Blood* 160ml MD1392 Anti-Foam Reagent* 300µl MD1431 MagneSil PMPs Fixed Yield* 25ml MD1451 Alcohol Wash, Blood* 120ml MD1412 Elution Buffer, Blood* 45ml MD1421 Wizard Magnetic 96 DNA Plant System (b) 2 x 96 preps FF x 96 preps FF3761 Wash Buffer, Plant 40ml A3811 MagneSil Genomic, Fixed Tissue System* 100 samples MD1490 MagneSil KF, Genomic System* 200 preps MD1460 MagneSil KF, Paramagnetic Particles* 40ml MD1471 Lysis Buffer, KF* 160ml MD1521 Wizard Magnetic DNA Purification System for Food (b) 200 preps FF preps FF3751 RNase A Solution, 4mg/ml* 1ml A7973 Lysis Buffer A, Food 100ml A8191 Lysis Buffer B, Food 100ml Z3191 Precipitation Solution, Food 150ml Z3201 Hard Ware Devices/Accessories Vac-Man Laboratory Vacuum Manifold, 1 each A sample capacity Vacuum Adapters 20 each A1331 Vac-Man 96 Vacuum Manifold 1 each A2291 Deep Well MagnaBot 96 Magnetic Separation Device* 1 each V3031 MagnaBot Spacer 1/8 inch 1 each V8581 Collection Plates (4-pack) 1 each A9161 Heat Transfer Block 1 each Z3271 MagnaBot 96 Magnetic Separation Device 1 each V8151 MagnaBot Spacer 1 each V8381 MagnaBot Adapter T1 1 each V8481 MagneSphere Technology Magnetic Separation Stand (two-position) 1.5ml Z5332 MagneSphere Technology Magnetic Separation Stand (twelve-position) 1.5ml Z5342 PolyATtract System 1000 Magnetic Separation Stand 1 each Z5410 *For Laboratory Use. (a) Australian Pat. No and other patents and patents pending. (b) U.S. Pat. Nos. 6,027,945, 6,368,800 and 6,673,631, Australian Pat. No and other patents and patents pending. Genomic DNA MagneBot, MagneSil, PolyATtract and Wizard are registered trademarks of Promega Corporation. ABI PRISM is a registered trademark of Applera Corporation. Biomek is a registered trademark of Beckman Coulter, Inc. 13

14 Information...always at your finger tips! Learn how ask Promega DNA Analysis Notebook Get the latest information to help you purify genomic DNA, amplify and clone your targets. RNA Analysis Notebook Isolate RNA and analyze by RT-PCR, microarrays and even RNAi. Call your local Promega representative to get your free copy or visit us on-line at: #BR129 #BR120 #BR152 14

15 Purify Plasmid DNA......at high yield, on any scale from manual to automated plate format. Promega plasmid DNA purification systems provide: Your choice of purification method: Promega provides a range of membrane filtration-based, magnetic particle-based and resin plasmid DNA purification systems. You choose the separation method best suited to your needs. Your choice of throughput: Both membrane-based and magnetic-based plasmid purification systems are available in manual and automated formats. Systems tailored to your purification scale: From miniprep to megaprep scale, there is a plasmid purification system for every need. Fully automated systems for high-throughput users: Automated methods and personalized automation support are available for all high throughput systems. Increased productivity: Save time and labor with our easy to use protocols. Purify Plasmid on Any Scale: Miniprep Midiprep Maxiprep Megaprep Multiwell-plate formats Full automation Plasmid DNA How can we help you achieve your goals today? A Comparison of Plasmid DNA Purification Systems. Culture Spin- Vacuum- Magnetic Manual Automated Volume Protocol Protocol Technology Handling Handling PureYield Plasmid Midiprep System (Cat.# A2492, A2495) Wizard Plus SV Minipreps (Cat.# A1330, A1340, A1460, A1470) Wizard SV 96 Plasmid System (Cat.# A2250, A2255) Wizard MagneSil Tfx System (Cat.# A2380, A2381) Wizard Plus Mini and Midipreps Systems (Cat.# A7100, A7500, A7510, A7640) Wizard Plus Maxi and Megapreps Systems (Cat.# A7270, A7300) Wizard PureFection System (Cat.# A2150, A2160, A2170) Wizard MagneSil Plasmid Purification System (Cat.# A1630, A1631) ml 1-10ml multiwell multiwell 1-100ml 100-1,000ml 5-125ml multiwell 15

16 The PureYield Plasmid Midiprep System Fast and reliable membrane-based column method for midiprep plasmid DNA purification. The PureYield Plasmid Midiprep System provides: Improve your productivity: Rapid protocol purifies large amounts of plasmid DNA in 30 minutes. Have confidence in your results: Highly purified and concentrated plasmid DNA gives proven performance in transfection in vitro expression and other molecular biology applications. Spend less time purifying DNA: High yield gives you more experiments per midiprep. Flexibility: The high binding capacity of PureYield columns allows plasmid purification from large culture volumes (250ml), providing maxiprep yield from a midiprep system. PureYield Plasmid Midiprep System 25 preps A preps A2495 For Research Use. PureYield System Done in 30 Minutes Wizard System 99 Minutes Qiagen Plasmid 200 Minutes 4587MB Firefly Luciferase (RLU) 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 Qiagen Eppendorf PureYield System Comparison of time required per midiprep using different systems. Each system protocol was performed according to the manufacturer s instructions using 50ml of an overnight culture of JM109 bacteria transformed with a high copy plasmid (pgem -3 plasmid). Total time to perform the midiprep is noted. DNA Preparation Methods 4701MA Comparison of transfection efficiency of purified plasmid DNA prepared using different midiprep systems. Comparison of transfection of plasmid DNA purified with the PureYield System and other midiprep systems. psicheck-2 Vector (Cat.# C8021), which carries a firefly luciferase gene, was isolated from E.coli using either the PureYield System, the HiSpeed Qiagen Plasmid Midi Kit or the Eppendorf Perfectprep Plasmid Midi Kit. HeLa cells were transfected using 0.07µg of DNA in a total of 25µl. The firefly luciferase signal was monitored with the Dual- Glo TM Luciferase Reporter System (Cat.# E1910). 4594CA Proper assembly of Lysate Clearing Column (blue) and DNA Binding Column (white) for use with the PureYield Plasmid Midiprep System vacuum-method purification protocol. 16

17 Wizard Plus SV Minipreps DNA Purification System Low-throughput, silica-membrane column-based miniprep plasmid DNA purification in manual format The Wizard Plus SV Minipreps System provides: High yield: Up to 20µg of plasmid from 10ml culture. Sequencing-quality DNA: The system is quality tested in fluorescent sequencing reactions to ensure read lengths of at least 500 base pairs with 98% accuracy. Time savings: Isolate 20 minipreps in less than 45 minutes. Compatibility with EndA+ strains: Incorporation of an alkaline protease step allows DNA isolation from problematic strains. Wizard Plus SV Minipreps DNA Purification System 50 preps A1330 Wizard Plus SV Minipreps DNA Purification System + Vacuum Adapters 50 preps A1340 Wizard Plus SV Minipreps DNA Purification System 250 preps A1460 Wizard Plus SV Minipreps DNA Purification System + Vacuum Adapters 250 preps A1470 For Laboratory Use. (a) U.S. Pat. No. 4,766,072. (b) U.S. Pat. No. 5,955,363. Plasmid DNA 1612TA09_6A M kb High-quality restriction digests using plasmid purified with the Wizard Plus SV Minipreps DNA Purification System. High-copy pgem -3Zf(+) Vector (a) (lanes 1 3) and low-copy palter -1 Vector (a,b) (lanes 4 6) were each digested in two separate Promega restriction enzyme reactions. All lanes show reproducible and efficient cutting of the plasmid DNA and are measured against the 1kb DNA Ladder (Cat.# G5711). pgem 3-Zf (+) plasmid sequenced with T7 sequencing primer using DNA purified by the Wizard Plus SV Minipreps DNA Purification System. 17

18 Wizard SV 96 Plasmid DNA Purification Systems High-throughput 96-well silica-membrane plate based plasmid DNA purification in automated or manual format The Wizard SV 96 System provides: Sequencing-quality DNA: Quality tested in fluorescent sequencing reactions to ensure read lengths of at least 600 base pairs with 98% accuracy. High quality at high speed: Process 96-wells of bacterial culture in 60 minutes or less, using a 96-well, vacuum filtration step to simultaneously clear bacterial cell lysates and bind DNA no centrifugation required! A walk-away, automated procedure: No manual intervention required once the E.coli cell pellets are placed on the robot deck. Hassle-free automated methods: Automated methods and support information available at: Ease-of-use and handling: The novel plate design prevents cross contamination during sample processing. Filtrate waste products are delivered directly to a vacuum trap, eliminating the need to empty waste collection vessels during the purification procedure. Wizard SV 96 Plasmid DNA Purification System 1 x 96 preps A x 96 preps A2255 Wizard SV 9600 Plasmid DNA Purification System* 100 x 96 preps A2258 Cat.# A2250, A2255 For Laboratory Use. *Cat.# A2258 is supplied with the components listed for Cat.# A2250 except for Alkaline Protease Solution, Nuclease-Free Water, 96-Well Deep Well Plates, DNA Elution Plates and Plate Sealers. Overnight cultures Lysate Clearing Plate Binding Plate Resuspend pelleted bacterial cells. Lyse. Neutralize. Transfer Lysates. Clear lysates. Bind DNA. Wash. The Wizard SV 96 System operates as a true walkaway system on automated purification platforms such as the Beckman Coulter Biomek FX laboratory automation workstation. It can also be used in a manual format. 3758CA06_2A Elute DNA. Elution Plate Wizard SV 96 is tested to ensure that purified DNA meets or exceeds 600 bases with 98% accuracy in fluorescent sequencing analysis. Highly pure plasmid DNA. 2651MB04_1A Wizard SV 96 Plasmid DNA Purification System protocol overview. Automated protocols are available at: 18

19 Wizard MagneSil Tfx System Transfection-quality plasmid DNA purification in a high-throughput 96-well miniprep format Wizard MagneSil Tfx System 4 x 96 preps A x 96 preps A2381 The Wizard MagneSil Tfx System provides: Lowered endotoxin plasmid DNA: Use of the endotoxin removal resin cuts endotoxin carryover as much as 95% over automated standard sequencing grade plasmid systems. Lower endotoxin levels produce better transfection efficiency. Improved mammalian protein expression: Three- to five-fold increase in protein expression compared to plasmid isolated from an automated sequencing-grade plasmid purification system. High-quality at high speed: Process 96-wells of bacterial culture in under 45 minutes with a Beckman Coulter Biomek FX, using a paramagnetic particle-based purification method for lysate clearing and DNA capture. Hassle-free automated methods: Automated method and support information available at: Clear Cell Debris Remove Endotoxin Bind DNA Plasmid DNA µg DNA Yields of Plasmid DNA Sample Number Sample-to-sample yield of plasmid DNA purified using the Wizard MagneSil Tfx System. Deepwell plates containing JM109 cells (4 O.D.600) transformed with pgl3 Control Vector DNA (a,b) (Cat.# E1741) were processed by three different users following the standard automated protocol provided in the Wizard MagneSil Tfx System Technical Bulletin #TB314. Each bar represents the average of 8 samples. Samples were quantitated by gel electrophoresis. 3905MA12_2A Wash Wash Bind DNA Elute Flow chart of Transfection-grade plasmid DNA purification using the Wizard MagneSil Tfx System. Dry 4387MA11_3A (a) U.S. Pat. No. 5,670,356. (b) The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,503,024, 5,674,713 and 5,700,673. A license (from Promega for research reagent products and from the Regents of the University of California for all other fields) is needed for any commercial sale of nucleic acid contined within or derived from this product. 19

20 Wizard Plus DNA Purification Systems Mini-, Midi-, Maxi- and Megaprep scale plasmid DNA purification in manual format The Wizard Plus Systems provide: Simplicity: Only a centrifuge, a vacuum source and a vacuum manifold required. Fast, manual format: Minipreps can be completed in as little as 15 minutes, Midipreps in 90 minutes, and Maxi- and Megapreps in 3 hours. Sequencing-quality DNA: Each Wizard Plus System is quality tested in fluorescent sequencing reactions to ensure read lengths of at least 500 base pairs with 98% accuracy. Safety and convenience: No phenol extraction or precipitation required. High yields: Using a high copy number plasmid, the following yields are obtained: 1 10µg DNA from 1 3ml culture; up to 200µg DNA from ml culture; up to 300µg 1mg from ml culture; and >1mg from 1,000ml culture. Wizard Plus Minipreps DNA Purification System 50 preps A preps A preps A7510 Wizard Plus Midipreps DNA Purification System 25 preps A7640 Wizard Plus Maxipreps DNA Purification System 10 preps A7270 Wizard Plus Megapreps DNA Purification System 5 preps A7300 Fluorescent dye primer sequencing of pgem Vector purified using the Wizard Plus Minipreps DNA Purification System. The system is tested to ensure that purified plasmid DNA can be sequenced at 98% accuracy over 500 bases using dye primer chemistry and an ABI PRISM 377 DNA sequencer. DNA purification using the Wizard Plus Systems requires use of a vacuum source and either a lab-assembled or commercial manifold such as Promega s Vac-Man Laboratory Vacuum Manifold (20-sample capacity). Plasmid Yield (µg) High Copy Number Low Copy Number 1460MA04_6A Culture Volume (ml) (a) U.S. Pat. No. 4,766,072. Plasmid DNA yield obtained using the Wizard Plus Midipreps DNA Purification System. Yield is shown as a function of culture volume and plasmid copy number. Representative yields of a high-copy number plasmid, pgem -3Zf(+) Vector (a), and a low-copy plasmid, pbr322, prepared in E. coli strain DH5α. 20

21 Wizard PureFection Plasmid DNA Purification System Low-throughput, transfection-grade plasmid DNA purification in scaleable manual format The Wizard PureFection System provides: Lower endotoxin plasmid DNA: Use of the endotoxin removal resin cuts endotoxin carryover as much as 95% over automated standard sequencing grade plasmid systems. Lower endotoxin levels produce better transfection efficiency. Safety and convenience: No phenol extraction or precipitation required, using a magnetic-based purification method for lysate clearing and DNA capture. High yield: 40µg DNA from 5ml culture; scale-up capacity up to ~1mg. Fast, efficient purification: The hand-held MagneSil Magnetic Separation Unit holds a 50ml tube and allows rapid and efficient capture of magnetic particles and their attached DNA. Scalable format: Rapid purification from a range of starting volumes from midiprep (5 25ml) to maxiprep (26 125ml) scale. Wizard PureFection Plasmid DNA Purification System 2 maxipreps/10 midipreps A maxipreps/25 midipreps A maxipreps/125 midipreps A2170 For Laboratory Use. Alkaline Lysis Produce cleared lysate. Selective Removal of Endotoxin with Magnet Endotoxin Removal Resin clears bacterial lysate of endotoxin. Plasmid DNA Bind Plasmid DNA Selective binding of plasmid DNA using MagneSil Paramagnetic Particles. 500 Endotoxin (EU/ml x 10 3 ) Wash & Elute Wash particles to remove salts and elute in water. Ethanol precipitate and resuspend DNA at desired concentration. 2347MB08_8A Endotoxin Removal Resin (mg) 2351MA08_8B Overview of Wizard PureFection Plasmid DNA Purification System protocol. Removal of endotoxin from bacterial cell lysates using Endotoxin Removal Resin. Over 95% of endotoxin is removed from bacterial lysates following a 5-minute incubation. 21

22 Wizard MagneSil Plasmid Purification System High-throughput 96-well plasmid DNA purification in an automated format The Wizard MagneSil Plasmid Purification System provides: A walk-away automated procedure: No manual intervention required once e. coli cell pellets are placed on robot deck. Sequencing-quality DNA: The system is quality tested in fluorescent sequencing reactions to ensure read lengths of at least 600 base pairs with 98% accuracy. High-throughput: Process multiple plates without user intervention. A simple, fast method: Plasmid DNA isolation is accomplished easily, with a simple, magnetic-based procedure, using a paramagnetic particle-based purification method for lysate clearing and DNA capture. Hassle-free automated methods: Automated methods and support information available at: Wizard MagneSil Plasmid Purification System 4 x 96 preps A x 96 preps A x 96 preps A1635 Average Yield (µg) Biomass O.D MB06_1A Capture MagneSil BLUE on MagnaBot Device. The Wizard MagneSil Plasmid Purification System is tested to ensure reliable DNA recovery. Plasmid DNA was purified from multiple samples grown to the stated O.D.600. DNA yield was measured on an agarose gel. Clear Cell Debris Wash Bind DNA Capture the MagneSil RED on the MagnaBot Device. Remove supernatant. Wizard MagneSil Plasmid Purification System protocol. Automated protocols are available at: Dry Elute 4388MA Accuracy (%) O.D. 4 O.D. 6 O.D. 8 O.D. 10 O.D. 12 O.D. MagneSil Paramagnetic Particles can be relied upon to purify high-quality template for fluorescent sequencing Bases Accuracy by read length for plasmid DNA sequenced following isolation by MagneSil Paramagnetic Particles. Results demonstrate greater than 700 consecutive bases analyzed with greater than 98% accuracy of base identity. 2890MA03_0A 22

23 Ordering Information PureYield Plasmid Midiprep System (a) 25 preps A preps A2495 Wizard Plus SV Minipreps DNA Purification System (b,c)* 50 preps A1330 Wizard Plus SV Minipreps DNA Purification System + Vacuum Adapters (b,c)* 50 preps A1340 Wizard Plus SV Minipreps DNA Purification System (b,c)* 250 preps A1460 Wizard Plus SV Minipreps DNA Purification System + Vacuum Adapters (b,c)* 250 preps A1470 Alkaline Protease Solution* 3ml A1441 Wizard SV 96 Plasmid DNA Purification System (b)* 1 x 96 preps A x 96 preps A2255 Wizard SV 9600 Plasmid DNA Purification System 100 x 96 preps A2258 Wizard SV Wash Solution* 185ml A1311 Wizard SV 96 Wash Solution 370ml A1318 Wizard SV 96 Neutralization Solution* 500ml A ml A1488 Wizard SV 96 Cell Resuspension Solution* 500ml A ml A7118 Wizard SV 96 Cell Lysis Solution* 500ml A ml A7128 Nuclease-Free Water* 150ml P1195 Alkaline Protease Solution* 3ml A1441 Wizard MagneSil Tfx System (a,d,e) 4 x 96 preps A x 96 preps A2381 Endotoxin Removal Resin 100ml A2191 4/40 Wash Solution 115ml A2221 Wizard Plus Minipreps DNA Purification System (f)* 50 preps A preps A preps A7510 Wizard Plus Midipreps DNA Purification System (f)* 25 preps A7640 Wizard Plus Maxipreps DNA Purification System (f)* 10 preps A7270 Wizard Plus Megapreps DNA Purification System (f)* 5 preps A7300 Wizard PureFection Plasmid DNA Purification System (a,d)* 2 maxipreps/ 10 midipreps A maxipreps/ 25 midipreps A maxipreps/ 125 midipreps A2170 Wizard MangeSil Plasmid Purification System (d,e) 4 x 96 preps A x 96 preps A x 96 preps A1635 MagneSil RED 100ml A1641 MagneSil BLUE 100ml A2201 Cell Resuspension Solution 500ml A7114 Cell Lysis Solution 500ml A7124 Neutralization Solution 500ml A7132 Elution Buffer 500ml A1655 Hard Ware Devices/Accessories Vac-Man Laboratory Vacuum Manifold, 20-sample capacity 1 each A7231 Vac-Man Jr. Laboratory Vacuum Manifold, 2-sample capacity 1 each A7660 Vacuum Adapters 20 each A1331 Vac-man 96 Vacuum Manifold 1 each A2291 Collar for Vac-Man 96 Vacuum Manifold 1 each A2311 Wizard SV 96 Binding Plates* 10 pack A each A2278 Wizard SV 96 Lysis Clearing Plates 10 pack A each A2248 MagnaBot 96 Magnetic Separation Device 1 each V8151 Collection Plates (4-pack) 1 each A9161 *For Laboratory Use. (a) U.S. Pat. No. 6,194,562, Australian Pat. No , Canadian Pat. No. 2,329,067 and other patents pending. (b) U.S. Pat. No. 5,981,235, Australian Pat. No and other patents pending. (c) Australian Pat. No and other patents and patents pending. (d) U.S. Pat. Nos. 6,027,945, 6,368,800 and 6,673,631, Australian Pat. No and other patents and patents pending. (e) U.S. Pat. No. 6,284,470 and other patents pending. (f) U.S. Pat. Nos. 5,658,548 and 5,808,041, Australian Pat. No and other patents and patents pending. MagneSil, palter, pgem, Vac-Man and Wizard are registered trademarks of Promega Corporation. Plasmid DNA 23

24 Superior Technical Services Scientists helping scientists to be successful! Get the best-in-class help and support you need from Promega. All Promega Technical Services Scientists have extensive laboratory experience. All have life science degrees and most have post-graduate or post-doc lab experience. If you have a question about one of our products, or even about a product that is not ours, feel free to contact us. Visit our web site for 24-hour a day support. Find online protocols, product FAQs, application information, references, publications, calculators and many other valuable support tools. It s all easy to access and easy to understand. Telephone Troubleshooting experiments Automation installation and support Global availability Multi-language capabilities Telephone: Toll Free in USA:

25 Purify DNA Fragments......from PCR, Sequencing Reactions and Gel Slices. Promega fragment DNA purification systems provide: High yield: Excellent recovery rates direct from PCR or from gels. Your choice of purification method: We provide a range of membrane filtration-based and magnetic particle-based systems. You choose the separation method best suited to your needs. Your choice of throughput: Both membrane-based and magnetic-based purification systems are available in manual and automated formats. Simplicity and sophistication: From manual, single-tube purification to full automation, there is a fragment purification system for every need. Fully automated systems for high-throughput users: Automated methods and personalized automation support are available for all high throughput systems. Increased productivity: Save time and labor from easy to use protocols. Experience you can trust: Have confidence in a supplier with over 25 years experience providing molecular biology systems and reagents. Purify Fragments: Directly from PCR From gels From sequencing reactions From restriction digests Away from phosphatases, kinases, polymerases Fragment DNA How can we help you achieve your goals today? A Comparison of Fragment DNA Purification Systems. Sequencing PCR Gel Fragment Spin Vacuum Magnetic Manual Automated Clean-Up Clean-Up Isolation Protocol Protocol Technology Handling Handling Wizard SV Gel and PCR Clean-Up (Cat.# A9281, A9282) Wizard SV 96 PCR Clean-Up (Cat.# A9340, A9341, A9342) Wizard PCR Preps (Cat.# A7170, A2180) Wizard DNA Clean-Up (Cat.# A7280) Wizard MagneSil Sequencing Reaction Clean-Up (Cat.# A1830, A1831, A1832) Wizard MagneSil PCR Clean-Up (Cat.# A1930, A1931) 25

26 Wizard SV Gel and PCR Clean-Up System High recovery of concentrated DNA fragments over a broad size range. The Wizard SV Gel and PCR Clean-Up System provides: Speed: Purify DNA fragments or PCR products in as little as 20 minutes directly from PCR or from agarose gels in either TAE or TBE buffer. High yield: 70 95% recovery of DNA fragments (100bp 10kb) from gels and 85 95% recovery of PCR products (100bp 3.2kb). Recovery of PCR products >3.2kb is similar to that from agarose gels. An application-tested system: Tested to ensure performance of purified PCR products in fluorescent sequencing. Purified DNA routinely achieves 700bp with >98% accuracy. Safety and convenience: No phenol extraction or precipitation required. Gel Purification Excise gel slice and add equal volume of Membrane Binding Solution Direct PCR Product Purification Wizard SV Gel and PCR Clean-Up System 50 preps A preps A9282 For Laboratory Use. 3,000bp 1,000bp M 100bp 500bp 1,000bp 3,199bp U P U P U P U P U: Unpurified P: Purified Gel analysis of DNA fragments before and after gel extraction using the Wizard SV Gel and PCR Clean-Up System. DNA fragments of the sizes indicated were analyzed before (U; unpurified) and after (P; Purified) purification. The marker (M) used is the 1kb DNA Ladder (Cat.# G5711). Wizard SV Gel and PCR Clean-Up System: Percent recovery of various sized fragments from agarose gels. 3789TA07_2A Vacuum Vacuum Adapter (Cat.# A1331) C for 10 minutes to solubilize gel slice Transfer sample to SV Minicolumn Add equal volume of Membrane Binding Solution to PCR reaction Centrifuge Bind DNA DNA Fragment Size Percent Recovery 70bp 39% 85bp 55% 100bp 84% 500bp 89% 1,000bp 92% 3,199bp 95% 9,416bp 95% Vac-Man Laboratory Vacuum Manifold (Cat.# A7231) Wash, removing solution by centrifugation or vacuum Transfer spin column to a 1.5ml microcentrifuge tube (not provided) Centrifuge Wizard SV Gel and PCR Clean-Up System: Percent recovery of various sized fragments directly from PCR. DNA Fragment Size Percent Recovery 200bp 87% 500bp 91% 1,000bp 92% 1,500bp 92% 3,199bp 87% Elute DNA fragment or PCR product 3792MA07_2A Flow chart of DNA fragment gel purification or direct PCR product purification using the Wizard SV Gel and PCR Clean-Up System. 26

27 Wizard SV 96 PCR Clean-Up System Rapid high-throughput 96-well recovery of fragment DNA The Wizard SV 96 PCR Clean-Up System provides: Speed: Process a 96-well plate in 20 minutes. High yield: Average percent recovery for 500bp and 1,000bp fragments is >90%, unaffected by common PCR additives. Gel slice purification: The system can be used to purify DNA from gel slices, as well as directly from PCR. Safety and convenience: No phenol extraction or precipitation required. An application-tested system: Tested to ensure performance of purified PCR products in fluorescent sequencing and microarray applications. Hassle-free automated methods: Automated methods and support information available at: Binding Plate Mix PCR sample and Membrane Binding Solution in equal volumes. Apply to Binding Plate. Apply vacuum to bind PCR product to membrane. Wash. Wizard SV 96 PCR Clean-Up System 1 x 96 preps A x 96 preps A x 96 preps A x 96 preps A9345 For Laboratory Use. A. B. Percent Recovery 100bp 200bp 300bp 500bp 1,000bp M P U P U P U P U P U ,000 Size (bp) 3801TA08_2A 3801TB08_2A Fragment DNA Collection Plate Elute bound PCR product with Nuclease-Free Water. 3743MA06_2A Purification and recovery of PCR products. PCR fragments of 100, 200, 300, 500, and 1,000 base pairs were purified using the Wizard SV 96 PCR Clean-Up System on the Beckman Coulter Biomek 2000 robotic workstation. Panel A. Purified (P) and unpurified (U) fragments were separated on an ethidium bromide-stained, 2% agarose gel. Panel B. Percent recovery of purified PCR products was quantitated using a Hitachi FMBIO Fluorescent Scanner. Results show the mean and standard deviation for 6 purified fragments of each size. For small PCR fragments (<500bp), optimal recovery requires a 95% ethanol wash. For larger fragments (>500bp), optimal results are achieved using an 80% ethanol wash. Wizard SV 96 PCR Clean-Up System protocol overview. 27

28 Wizard PCR Preps DNA Purification System Reliable low-throughput PCR and agarose gel slice DNA fragment clean-up The Wizard PCR Preps DNA Purification System provides: High yield: 70 90% recovery of DNA fragments (500bp) from low-melting point agarose gels and 95 99% recovery of PCR products (300bp 1.5kb) directly from reactions. Efficiency: Process multiple samples simultaneously using the Vac-Man Laboratory Vacuum Manifold (20 sample capacity). Safety and convenience: No phenol extraction or precipitation required. Confidence: Removal of excess nucleotides, primers and small nonspecific amplification products such as primer-dimers from PCR. Wizard PCR Preps DNA Purification System 50 preps A preps A2180 For Laboratory Use bp Before Purification M M After Purification Purification and analysis of PCR products from low-melting agarose using the Wizard PCR Preps DNA Purification System. Lane 1, 250ng pgem DNA Markers; lane 2, β-1-anti-trypsin PCR product; lane 3, purified β-1-antitrypsin PCR fragment; lane 4, b-globin PCR reaction product; lane 5, purified b-globin PCR fragment. 0348TA08_3A 1, TA11_4A Recovery of PCR products using the Wizard PCR Preps Resin. A representative sample from the simultaneous purification of 96 PCR reactions was chosen to determine the effectiveness of the DNA purification process. Equivalent amounts from before and after purification were separated on a 1% agarose gel and stained with ethidium bromide. 28

29 Wizard DNA Clean-Up System Safe, easy clean-up of linear or circular DNA The Wizard DNA Clean-Up System provides: Speed: Purify DNA in as little as 15 minutes. Simple, effective purification: Purify linear and circular DNA (200bp 50kb) away from contaminants such as restriction enzymes, phosphatases, kinases, DNA polymerases, exonucleases and endonucleases (including DNase I but not RNases), mononucleotides and salts. Efficiency: Process multiple samples simultaneously using the Vac-Man Laboratory Vacuum Manifold (20 sample capacity). Safety and convenience: No phenol extraction or precipitation required. Applications tested: Eluted DNA demonstrated for use in cloning, clean-up of hi-sulfate treated genomic DNA and other applications. Wizard DNA Clean-Up System 100 preps A7280 For Laboratory Use. Removal of Common Impurities by the Wizard DNA Clean-Up System. Contaminant Restriction enzymes Heat-stable restriction enzymes Phosphatases Kinases Ligases DNA polymerases Taq DNA polymerases Exonucleases DNase I RNases Mononucleotides Salts Oligonucleotides Removed Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes No Fragment DNA 29

30 Wizard MagneSil Sequencing Reaction Clean-Up System High-throughput automated 96-well or 384-well sequencing reaction clean-up made simple and economical The Wizard MagneSil Plasmid Purification System provides: High-throughput: Process multiple 96-well or 384-well plates without user intervention. A simple, fast method: DNA isolation is accomplished easily, with a simple, magnetic-based procedure. High quality purified DNA: Typical Phred 20 values of bases. 98% accuracy to at least 600 bases. Efficiency: Save labor with a walk-away high-throughput sequencing reaction clean-up automated method. Hassle-free automated methods: Automated methods and support information available at: Sequencing Reaction Clean-Up in 96-Well Plates Wizard MagneSil Sequencing Reaction Clean-Up System 1 x 96 preps A x 96 preps A x 96 preps A x 96 preps A1835 Sequencing Reaction Clean-Up in 384-Well Plates MagneSil GREEN 100ml A8231 MagnaBot 384 Magnetic Separation Device 1 each V Well Plate, Conical 10/pack V5311 Cat.# A1830, A1831, A1832, A1835, A8231, V8241 For Laboratory Use CA_1A # of Samples Clean-up is performed using the MagnaBot II Magnetic Separation Device (Cat.# V8351) accompanied by the Plate Clamp 96 (Cat.# V8251). These devices are designed to work with most robotic platforms Phred 20 Read Length (bp) Phred 20 values for sequencing data from each well of a 96-well plate using the Wizard MagneSil Sequencing Reaction Clean-Up System. Phred is a base-calling program that assigns an error probability to each base. A phred value of 20 for a given base assigns an error probability of 1 in a 100 for that base. 3271MA02_1B MagnaBot 384 Magnetic Separation Device (Cat.# V8241). The MagnaBot 384 is constructed such that the magnetic field draws particles to the corners of the processing plate. 3995CA02_3A Wizard MagneSil Sequencing Reaction Clean-Up System undergoes rigorous testing for performance. DNA prepared by this method routinely achieves 98% accuracy out to 600 bases. 30

31 Sequencing Reaction Add MagneSil GREEN Add Ethanol Wash Solution Add Sequencing Gel Loading Solution Place on MagnaBot II Place on MagnaBot II Place on MagnaBot II Remove Supernatant Remove Supernatant Transfer eluate to 96-well plate for sequence analysis Binding Washing Elution Overview of the Wizard MagneSil Sequencing Reaction Clean-Up System protocol. 3169MB04_1A Fragment DNA A. MagneSil PMPs B. EtOH Precipitation 3250TA02_1A A comparison of electropherograms of purified sequencing reactions analyzed on an ABI PRISM 3700 DNA Sequencer. Panel A: Reactions purified with the Wizard MagneSil Sequencing Reaction Clean-Up System. Panel B: Reactions purified by ethanol precipitation. 31

32 Wizard MagneSil PCR Clean-Up System High-throughput PCR clean-up ideal for 96-well or 384-well automated format The Wizard MagneSil PCR Clean-Up System provides: High-throughput: Process multiple 96-well or 384-well plates without user intervention using a magnetic-based purification method for DNA capture that is ideally suited for automation. A simple, fast method: No centrifugation or vacuum manifold required. High quality DNA: Primer removal is consistently >98% with purification of PCR fragments from 150bp 23kb in size. An application-tested system: Tested to ensure performance of purified PCR products in fluorescent sequencing and microarray applications. Hassle-free automated methods: Automated methods and support information available at: PCR Clean-Up in 96-Well Plates Wizard MagneSil PCR Clean-Up System (a) 4 x 96 preps A x 96 preps A x 96 preps A1935 PCR Clean-Up in 384-Well Plates MagneSil YELLOW 100ml A9231 Wash Solution 500ml A8241 MagnaBot 384 Magnetic Separation Device 1 each V Well Plate, Flat 10/pack V5291 Cat.# A1930, A1931, A1935, A8241, A9231, V8241 For Laboratory Use. 100 % Recovery MagnaBot 96 Magnetic Separation Device (Cat.# V8151). 3993CA02_3A ,000 PCR Product Size (bp) 1, MA08_2A Recovery of PCR products of varying size using the Wizard MagneSil PCR Clean-Up System. A range of sizes of PCR products, generated using PCR Master Mix (a) (Cat.# M7505), were recovered using the Wizard MagneSil PCR Clean-Up System on a liquid-handler. The yield of each PCR product was determined by electrophoresis on a 2% agarose gel stained with ethidum bromide. The mean and standard deviation of four separate determinations for each PCR product are shown. MagnaBot 384 Magnetic Separation Device (Cat.# V8241). The MagnaBot 384 is constructed such that magnetic field draws the particles to the corners of the wells of the processing plate. 3995CA02_3A (a) The PCR process is covered by patents issued and applicable in certain countries*. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. *In the U.S., effective March 29, 2005, U.S. Pat. Nos. 4,683,195, 4,965,188 and 4,683,202 will expire. In Europe, effective March 28, 2006, European Pat. Nos. 201,184 and 200,362 will expire. 32

33 PCR Reactions Add Wash Solution Add MagneSil YELLOW Place on MagnaBot 96 Device Add Nuclease-Free Water Place on MagnaBot 96 Device Remove Supernatant Place on MagnaBot 96 Device Remove Supernatant Transfer eluate to 96-well plate. DNA Binding Repeat wash cycle 2X with 80% ethanol. Washing Overview of the Wizard MagneSil PCR Clean-Up System protocol. Elution of DNA 3288MB03_1A Fragment DNA bp M Unpurified 100% 50% 25% 12.5% 6.25% Purified 1, TA07_1A The Wizard MagneSil PCR Clean-Up System removes primer-dimers from PCR products with high yield and consistent results. A titration of unpurified prothrombin PCR product is shown (100%, 50%, 25%, 12.5% and 6.25% of the PCR sample). The ~100bp primer-dimers are visible in these lanes. Eight replicates purified using the Wizard MagneSil PCR Clean-Up System show the 506bp prothrombin PCR product with no visible primer-dimers. The ethidium bromide-stained 1% agarose gel was imaged using an Alpha Innotech FluorChem fluorescence imaging system. Lane M, 100bp DNA Ladder (Cat.# G2101). 33

34 Ordering Information Wizard SV Gel and PCR Clean-Up System* 50 preps A preps A9282 Wizard SV 96 PCR Clean-Up System (a) * 1 x 96 preps A x 96 preps A x 96 preps A x 96 preps A9345 Membrane Binding Solution* 20ml A9301 Wizard PCR Preps DNA Purification System (b) * 50 preps A preps A2180 Wizard DNA Clean-Up System (c) * 100 preps A7280 Wizard MagneSil Sequencing Reaction Clean-Up System (d) * 1 x 96 preps A x 96 preps A x 96 preps A x 96 preps A1835 MagneSil GREEN* 100ml A8231 Wizard MagneSil PCR Clean-Up System (d) * 4 x 96 preps A x 96 preps A x 96 preps A1935 MagneSil YELLOW* 100ml A9231 Wash Solution* 500ml A8241 Nuclease-Free Water* 150ml P1195 Hard Ware Devices/Accessories Vac-Man 96 Vacuum Manifold 1 each A2291 Vacuum Adapters 20 each A1331 Wizard SV 96 Binding Plates* 10 pack A each A2278 MagnaBot II Magnetic Separation Device 1 each V8351 Plate Clamp 96 1 each V8251 Plate Stand 1 each V8261 MagnaBot 96 Magnetic Separation Device 1 each V8151 Plate Clamp 96 1 each V8251 Plate Stand 1 each V8261 MagnaBot Spacer 1 each V8381 MagnaBot Adapter T1 1 each V8481 Collection Plates (4-pack) 1 each A9161 MagnaBot 384 Magnetic Separation Device* 1 each V Well Plate, Conical 10 pack V Well Plate, Flat 10 pack V5291 *For Laboratory Use. (a) Australian Pat. No and other patents and patents pending. (b) Licensed under U.S. Pat. No. 5,075,430. (c) U.S. Pat. Nos. 5,658,548 and 5,808,041, Australian Pat. No and other patents and patents pending. (d) U.S. Pat. Nos. 6,027,945 and 6,368,800, Australian Pat. No and other patents and patents pending. MagnaBot, MagneSil, pgem and Wizard are registered trademarks of Promega Corporation. Dual-Glo is a trademark of Promega Corporation. Biomek is a registered trademark of Beckman Coulter, Inc. FMBIO is a registered trademark of Hitachi Software Engineering Company, Ltd. ABI PRISM is a registered trademark of Applera Corporation. FluorChem is a registered trademark of Alpha Innotech Corporation. HiSpeed is a trademark of Qiagen. Perfectprep is a registered trademark of Eppendorf. 34

35 Isolate RNA......from a variety of sample materials, on a scale of your choosing. Promega s Total RNA and mrna Isolation Systems provide: Your choice of purification method: Promega provides a range of solution, membrane filtration-based and magnetic particle-based systems. You choose the separation method best suited to your needs. Your choice of throughput: Both membrane-based and magnetic-based RNA isolation systems are available in manual and automated formats. Systems tailored to your purification scale: Mini-isolation from small sample sizes to scaleable, solutions-based formats, there is a total RNA purification system for every need. Direct isolation of total RNA or mrna from biological materials: Systems are available for direct isolation from a variety of starting materials. Increased productivity: Save time and labor with our easy to use protocols. Purify RNA From: Blood Tissue Cultured Cells Plant Material Bacteria How can we help you achieve your goals today? A Comparison of RNA Isolation Systems. Total RNA Isolation Systems RNAgents Total RNA Isolation System (Cat.# Z5110) Total Animal Culture Plant Manual Automated Magnetic Spin Vacuum RNA Blood Bacteria Tissue Cells Material Handling Handling Technology Protocol Protocol Total RNA SV Total RNA Isolation System (Cat.# Z3100, Z3105) SV 96 Total RNA Isolation System (Cat.# Z3500, Z3505) MagneSil Total RNA mini-isolation System (Cat.# Z3351) mrna Isolation Systems PolyATtract Automated System (Cat.# Z5671) PolyATtract System 1000 (Cat.# Z5400, Z5420) PolyATtract mrna Isolation Systems (Cat.# Z5200, Z5300) 35

36 RNAgents Total RNA Isolation System Scalable solution-based total RNA purification for maximum flexibility RNAgents Total RNA Isolation System Scalable Z5110 For Laboratory Use. The RNAgents Total RNA Isolation System provides: Speed: The solution-based procedure can be completed in 90 minutes to 3 hours. Scalable: Isolate RNA from as little as 5mg, as much as 6g of tissue, or from cultured cells. Simplicity: A solution-based protocol for RNA isolation that can be performed with ease in any laboratory setting. Performance: Yield and purity for today s demanding applications (e.g. microarrays and qrt-pcr) Complete system: Purity total RNA from blood, tissue cells, plant leaf, etc. Maiximum flexibility: Denaturing Solution available separately for maximum flexibility, allowing you the option to supply the other system components (phenol/chloroform, isoamyl alcohol and isopropanol). Average Yields of Total RNA from a Variety of Tissues. Tissue RNAgents Total RNA Yield HeLa cells 1.6mg RNA/10 8 cells Human white blood cells 1.3mg RNA/10 8 cells Mouse intestine 2.3mg RNA/g tissue Mouse spleen 8.3mg RNA/g tissue Mouse lung 1.9mg RNA/g tissue Mouse kidney 3.1mg RNA/g tissue Mouse liver 6.6mg RNA/g tissue E. coli Control Array Cy 3 Cy TA11_1A B. Jurkat Control Array 28S Cy 3: β-actin Cy 5: Kan 18S Analysis of RNA isolated from various tissues with the RNAgents System. RNA was isolated from the following sources: HeLa cells (lane 1), mouse intestine (lane 2), mouse spleen (lane 3), mouse lung (lane 4), mouse kidney (lane 5) and mouse liver (lane 6). Five micrograms of each sample was resolved on a 1% denaturing agarose gel (2.2M formaldehyde). 0349TA11_5A Hybridization of fluorescent cdna targets to E.Coli and Jurkat Cell Arrays. Panel A: Hybridization results for the E. coli control array. One array was hybridized with a Cy 3 target and another with a Cy 5 target. Panel B: Hybridization results for the Jurkat Control Array. Fluorescent Cy 3 and Cy 5 targets were co-hybridized to the same array, demonstrating specificity for their complementary sequences. 36

37 SV Total RNA Isolation System Simple and reliable total RNA purification from tissues, cultured cells or white blood cells The SV Total RNA Isolation System provides: Speed: The procedure can be completed in 60 minutes. Safety and convenience: Silica membrane based, no phenol extraction required. Confidence in your results: Includes a DNase I treatment step to ensure removal of genomic DNA. No detectable DNase I carryover to the final RNA preparation. Peace of mind: Only system that includes DNase treatment step in the standard protocol. Applications tested RNA: RNA suitable for all routine molecular biology applications, including RT-PCR and Northern blotting. Average Yields of Total RNA Using SV Total RNA Isolation System. SV Total RNA Isolation System 10 preps Z preps Z preps Z3105 For Laboratory Use. bp 1,500 1, β-actin APC M 5µl 5µl 10µl 15µl RNA isolated from 1ml of human blood using the SV Total RNA Isolation System. RT-PCR was performed using the indicated volumes of eluted RNA and primers complementary to human β-actin or human Adenomatous Polyposis Coli (APC) gene with the Access RT-PCR System (a,b) (Cat.# A1280, A1250). Maximum Avg. Yield Avg. Yield Amount to per Prep per mg Samples Process (µg) Tissue (µg) A 260 /A 280 Mouse Tissues Liver 30mg Kidney 20mg Spleen 15mg Brain 60mg Muscle 30mg Rat Tissues Pancreas 30mg Heart 60mg Lung 60mg Bacteria E. coli 1 x 10 9 cells 36 N/A 2.0 Yeast S. cerevisiae 4 x 10 7 cells 19 N/A 2.1 Plant Tomato Leaf 30mg Cell Line RAW x 10 6 cells 51 N/A 2.1 The vacuum protocol for the SV Total RNA Isolation System requires use of the Vacuum Adapters and a vacuum manifold such as Promega s Vac-Man Laboratory Vacuum Manifold (20-sample capacity). Total RNA N/A = Not Applicable. Average yield is that of RNA only. Other commercial systems may appear to give higher yields, but this is frequently due to DNA carryover. 37

38 A. Homogenize samples (tissues, cultured cells or white blood cells) in SV RNA Lysis Buffer. Transfer to fresh tube and add SV RNA Dilution Buffer; mix and centrifuge. Transfer to fresh tube; add 95% Ethanol and mix. Spin Centrifuge for one minute. Wash with SV RNA Wash Solution. DNase Treatment. Add SV DNase Stop Solution. Centrifuge for one minute. Wash 2X with SV RNA Wash Solution. Elute RNA into Elution Tube. Transfer to Spin Column Assembly. Vacuum Remove lysate by vacuum. Wash with SV RNA Wash Solution. DNase Treatment. Add SV DNase Stop Solution. Wash 2X with SV RNA Wash Solution. Remove Spin Basket and place in collection tube. Centrifuge. Place Spin Basket in Elution Tube. B. Interslide CV (%) Exp. 1 Exp. 2 Exp. 3 High degree of experimental reproducibility using RNA purified by the SV Total RNA Isolation System. Panel A. Subgrids from four representative 4K human cancer arrays (provided by Corning Incorporated) are shown. Labeled cdna was generated with the ChipShot components of the Pronto! Plus System using 5ug of total RNA purified using the SV Total RNA System. Panel B. Reproducibility was assessed by determining the %CV for 4000 features from 3-4 arrays. The inter-slide %CV was <10% indicating a high degree of reproducibility within and between experiments as a result of high quality input total RNA. 4413TA12_3A Total time: minutes Schematic of the SV Total RNA Isolation System protocol. 1 Promega Competitor M High-quality RNA from the SV Total RNA isolation system ensures success in subsequent experiments such as microarrays. (a) The PCR process is covered by patents issued and applicable in certain countries*. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license. *In the U.S., effective March 29, 2005, U.S. Pat. Nos. 4,683,195, 4,965,188 and 4,683,202 will expire. In Europe, effective March 28, 2006, European Pat. Nos. 201,184 and 200,362 will expire. (b) U.S. Pat. Nos. 4,966,964, 5,019,556 and 5,266,687, Australian Pat. Nos. 616,881 and 641,261 and other pending and issued patents, which claim vectors encoding a portion of human placental ribonuclease inhibitor, are exclusively licensed to Promega Corporation. Formaldehyde gel electrophoresis of RNA. RNA was isolated from 30mg of liver tissue following the SV Total RNA Isolation System protocol (lanes 1 6) and a competitors protocol (lanes 7 12). Ten percent (10µl) of the final prep volume of 100µl for each sample was analyzed by electrophoresis in a 1% MOPS-EDTAformaldehyde agarose gel and visualized by including 0.1µg/µl ethidium bromide in the sample loading buffer. Promega s system consistently yielded an average of 100µg. The competitor s system yielded an average of 70µg with wide variability. 1906TA09_7A 38

39 SV 96 Total RNA Isolation System High-throughput 96-well total RNA purification The SV 96 Total RNA Isolation System provides: Efficiency: Save labor with a walk-away high-throughput 96-well purification automated method. 96-samples processed is less than 1 hour. A unique design: The novel vacuum manifold eliminates waste handling, and the novel plate design prevents cross-contamination during sample processing. Confidence in your results: A DNase I treatment step is included in the protocol to ensure removal of genomic DNA contamination. There is no detectable DNase carryover into the final RNA preparation. An applications-tested system: Tested to ensure purified RNA performs optimally in RT-PCR. Hassle-free automated methods: Automated methods and support information available at: SV 96 Total RNA Isolation System 1 x 96 each Z x 96 each Z3505 For Laboratory Use. 3446CA06_1A Automated isolation of total RNA using the SV 96 Total RNA Isolation System on the Beckman Coulter Biomek FX SV 96 SV µg RNA HeLa NIH3T3 CHO 3467MB07_1A Total RNA The SV 96 Total RNA Isolation System provides consistent and reliable recovery of high quality total RNA. Panel A: RNA yields from several cell lines using 1 x 10 5 cells is consistently 0.3µg or more. Total RNA can be purified from 96 samples at once in less than an hour. 39

40 MagneSil Total RNA mini-isolation System High-throughput 96-well or 384-well purification of highly concentrated total RNA The MagneSil Total RNA mini-isolation System provides: A simple, fast method: Elution volumes as low as 15µl give concentrated RNA. Only 30 minutes to process one 96-well plate and 50 minutes to process a single 384-well plate on a Beckman Coulter Biomek FX laboratory workstation. Confidence in your results: A DNase I treatment step is included in the protocol to ensure removal of genomic DNA contamination. A performance-tested system: RNA rigorously tested in end-point RT-PCR and quantitative, real-time RT-PCR. Efficiency: Save labor with a walk-away high-throughput 96-well purification automated method. 96-samples processed in only 30 minutes. Designed for screening protocols: Total RNA isolation from small sample sizes (up to 10 5 cultured cells, 20mg tissue or 20µl whole blood/well in a 96-well format, and up to 10 3 cultured cells or 5µl whole blood/well in a 384-well format). Hassle-free automated methods: Automated methods and support information available at: MagneSil Total RNA mini-isolation System 4 plates Z3351 For Laboratory Use. Add sample lysate to MagneSil RNA PMPs. Mix. Capture PMPs. Discard supernatant. Wash PMPs. Incubate PMPs with DNase 10 minutes. Add DNase Stop Solution. Capture PMPs. Discard supernatant. Wash PMPs. Elute RNA. 4316MB11_3A Overview of the MagneSil Total RNA mini-isolation System protocol. 40

41 µg Yield A. Ct GAPDH MagneSil RNA 60 Concentration silica membrane ,000 Cell Number 10, ,000 1E ng/µl B c-myc 10 0 MagneSil RNA silica membrane 4376MA11_3A Ct Yield of total RNA from HeLa cells using the MagneSil Total RNA mini-isolation System. Total RNA was isolated from 1 x 10 5 HeLa cells/well using either the MagneSil Total RNA mini-isolation System or a silica membrane based purification method. Total RNA concentration and yield were calculated using RiboGreen RNA Quantitation Reagent (Molecular Probes) ,000 Cell Number 10, ,000 1E TC Concentration (ng/µl) Real-time RT-PCR analysis of RNA purified using the MagneSil Total RNA mini- Isolation System. To measure linearity and sensitivity of RNA purification, GAPDH (abundant mrna) and c-myc (rare mrna) were amplified in real-time RT-PCR from RNA purified from various titrations of HeLa cells. Twenty microliter aliquots of total RNA isolated from a dilution series of HeLa cells in a 96-well plate were used as template in 100µl reverse transcription reactions. Panel A: Five microliter aliquots (n=3) of the RT reaction were used for PCR of a GAPDH target. Panel B: Five microliter aliquots (n=1) of the RT reaction were used for PCR of a c-myc target. GAPDH signal and c-myc signal were detected from as little as 10 cells. 0.6 Elution Volume (µl) Total RNA Yield (µg) Elution Volume (µl) 4377MA11_3A MagneSil Total RNA mini-isolation System: titration of elution volume. Total RNA was isolated from 2 x 10 4 HeLa cells/well using the MagneSil Total RNA mini-isolation System, and eluted in various volumes from 50µl down to 10µl. Yield and concentration obtained for each elution volume are shown. Total RNA concentration was calculated using RiboGreen RNA Quantitation Reagent (Molecular Probes). 41

42 PolyATtract Automated System High-throughput, mrna purification for sensitive applications The PolyATtract Automated System provides: Your choice of starting material: Linear recovery of poly(a)+ RNA from µg of eukaryotic total RNA. Alternatively, work with mg of eukaryotic tissue in lysate or of cultured mammalian cells. Strong, selective mrna binding: A magnetic-based purification method for mrna capture that is based on hybridization of poly(a)+ mrna to a biotinylated oligo(dt) primer. The bound mrna is then captured by streptavidn-coated paramagnetic particles. Removal of >99% of ribosomal RNA as judged by quantitative, real-time RT-PCR. Confidence with expression profiles: Expression profiles remain the same as samples from total RNA and poly(a)+ RNA. Freedom from well-to-well cross-contamination: No detectable well-to-well contamination as judged by RT-PCR. Efficiency: Save labor with walk-away automation of mrna purification directly from cultured cells, tissue lysates or purified total RNA. Hassle-free automated methods: Automated methods and support information available at PolyATtract Automated System 4 x 96 Z5671 For Laboratory Use. A B GAPDH Target Cycle Total RNA (Avg. Ct = 23.0) mrna From Total RNA (Avg. Ct = 24.9) mrna From Cells (Avg. Ct = 23.8) S rrna Target Cycle 1:10 Total RNA 1:100 Total RNA mrna From Total RNA mrna From Cells 1:1,000 Total RNA 1:10,000 Total RNA 4073TA03_3A Detection of Ten copies of GAPDH and c-myc using real-time RT-PCR analysis of RNA purified using the MagneSil Total RNA Mini-Isolation System. To measure linearity and sensitivity of RNA purification, GAPDH (abundant mrna) and c-myc (rare mrna) were amplified in real-time RT-PCR from RNA purified form various titrations of HeLa cells. Twenty microliter aliquots of total RNA isolated from a dilution series of HeLa cells in a 96-well plate were used as template in 100_l reverse transcription reactions. Panel A: Five microliters aliquots (n=3) of the RT reaction were used for PCR of a GAPDH target. Panel B: Five microliter aliquots (n=1) of the RT reaction were used for PCR of a c-myc target. 42

43 PolyATtract mrna Isolation Systems Scalable, flexible mrna purification for all applications The PolyATtract mrna System provides: Speed: mrna isolation is completed in 45 minutes or less. Strong, selective mrna binding: A magnetic-based purification method for mrna capture that is based on hybridization of poly(a)+ mrna to a biotinylated oligo(dt) primer. The bound mrna is then captured by streptavidin-coated paramagnetic particles. Removal of >99% of ribosomal RNA as judged by quatitative, real-time RT-PCR. Your choice of starting material: Configurations available for large or small amounts of cells or tissues. mrna isolation from total RNA in low throughput format (PolyATtract Systems I-IV) or directly from crude cell or tissue lysates in scaleable format (PolyATtract System 1000). Convenience and ease-of-use: mrna isolation accomplished easily, with a simple, magnetic-based procedure. Applications tested: Highly pure mrna suitable for molecular biology applications such as in vitro translation, cdna synthesis, PCR, RNase protection, primer extension and Northern blots. PolyATtract System 1000 with Magnetic Stand Scalable Z5420 PolyATtract System 1000 without Magnetic Stand Scalable Z5400 PolyATtract mrna Isolation System II with Magnetic Stand 3 isolations Z5200 PolyATtract mrna Isolation System I (Refill for Z5200) 3 isolations Z5210 PolyATtract mrna Isolation System III with Magnetic Stand 15 isolations Z5300 PolyATtract mrna Isolation System IV (Refill for Z5300) 15 isolations Z5310 For Laboratory Use. Average Yields of Poly(A)+ RNA Using PolyATtract System Size of Yield of µg mrna/ Sample Sample mrna A 260 /A 280 g sample Liver 1,000mg 220µg Kidney 420mg 138µg Spleen 390mg 282µg Pancreas 400mg 148µg Lung 160mg 16µg NIH3T cells 20µg Total RNA (a) U.S. Pat. No. 5,693,784. (b) U.S. Pat. No. 6,218,531, Australian Pat. No and other patents pending. 43

44 Ordering Information RNAgents Total RNA Isolation System* Scalable Z5110 RNAgents Denaturing Solution* 120ml Z5651 SV Total RNA Isolation System (a,b) * 10 preps Z preps Z preps Z3105 SV RNA Red Blood Cell Lysis Solution* 200ml Z3141 SV RNA Lysis Buffer* 50ml Z3051 SV 96 Total RNA Isolation System (a) * 1 x 96 each Z x 96 each Z3505 SV RNA Lysis Buffer* 50ml Z3051 SV RNA Wash Solution* (concentrated) 58.8ml Z3091 Nuclease-Free Water* 150ml P1195 Hard Ware Devices/Accessories Vac-Man Laboratory Vacuum Manifold, 20-sample capacity 1 each A7231 Vacuum Adapters 20 each A1331 Vac-Man 96 Vacuum Manifold 1 each A2291 Wizard SV 96 Binding Plates 10 pack A each A2278 MagneSil Magnetic Separation Unit 1 each A2231 MagnaBot 96 Magnetic Separation Device 1 each V8151 Heat Transfer Block 1 each Z3271 MagnaBot Spacer 1/8 inch 1 each V8581 MagnaBot Spacer 1 each V8381 1/4 inch Foam Spacer 1 each Z3301 PolyATtract System 1000 Magnetic Separation Stand 1 each Z5410 Cat.# P1195, Z5261, Z5482, Z5531 Cat.# A2271, A2278, Z3051, Z3091, P1195. For Laboratory Use. (a) U.S. Pat. No. 6,218,531, Australian Pat. No and other patents pending. (b) Australian Pat. No and other patents and patents pending. MagneSil Total RNA mini-isolation System (a) * 4 plates Z3351 PolyATtract Automated System* 4 x 96 Z5671 Nuclease-Free Water* 150ml P1195 Biotinylated Oligo(dT) Probe (50pmol/µl)* 35µl Z5261 Steptavidin MagneSphere Paramagnetic Particles* 25ml Z5482 PolyATtract GTC Extraction Buffer* 120ml Z5531 (c) U.S. Pat. No. 5,693,784. MagneSil is a registered trademark of Promega Corporation. ChipShot and Pronto! are trademarks of Promega Corporation. Cy is a registered trademark of Amersham Biosciences Ltd. Biomek is a registered trademark of Beckman Coulter, Inc. RiboGreen is a registered trademark of Molecular Probes, Inc. PolyATtract System 1000 with Magnetic Stand (c) * Scalable Z5420 PolyATtract System 1000 without Magnetic Stand (c) * Scalable Z5400 PolyATtract mrna Isolation System II with with Magnetic Stand* 3 isolations Z5200 PolyATtract mrna Isolation System I* (Refill for Z5200) 3 isolations Z5210 PolyATtract mrna Isolation System III with with Mangetic Stand* 15 isolations Z5300 PolyATtract mrna Isolation System IV* (Refill for Z5300) 15 isolations Z5310 Promega Corporation 2800 Woods Hollow Road Madison, WI USA Telephone Fax Promega Corporation. All Rights Reserved. Prices and specifications subject to change without prior notice. Printed in USA 09/ BR-PU Part #BR106 44