integrated solutions for nucleic acid...

for Nucleic

Acid

Purification

Integrated Solutions

2

Genomic DNA 3

Plasmid DNA 15

Fragment DNA 25

Total RNA 35

Table of Contents

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 Systemsfrom scientists for scientists, for all

your perfomance and throughput needs.

3

Geno

mic

DNA

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)

How can we help you achieve your goals today?

Sample types: • Blood

• Bacteria

• Mouse tail and animal tissue

• Tissue culture cells

• Plants

• Food

• Applications for other sample types

Purify High-Quality DNA...

A Comparison of Genomic DNA Purification Systems.

...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 plateor automated plate/tube) to meet your requirements.

• Convenience: Choose a kit that processes multiple sample types or forchallenging samples, a specific sample-type kit.

4

High molecular weight gDNA from multiple sampletypes 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 foramplification, restriction enzyme digestion, and southern blottingfrom 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 inabout 1 hour.

DNA Yields from Various Starting Materials.

Amount of TypicalSource Starting Material DNA YieldWhole Blood 300µl 5–15µg

3ml 25–50µg10ml 250–500µg

96-well plate, 50µl/well 0.2–0.7µg

Tissue Culture Cells 106–107 cells 5–30µg

Animal TissueMouse Liver 11mg 15–20µgMouse Tail 0.5–1cm of tail 10–30µg

Insect Cells 5 × 106 cells 16µg

Plant Leaf Tissue 40mg 7–12µg

Bacterial Culture* 108–1010 cells 5–20µg

Yeast* 1.9 × 108 cells 4.5–6.5µg

*Overnight culture.

bp10,000 –3,000 –1,000 –

M 1 2

+ 300µl 100µl 50µl 20µl

3 4 5 6 7 8 9 10

2862

TB

02_0

A

Sample

Transfer supernatantto new tube containingisopropanol.

Centrifuge.

Aspirate ethanol.Air-dry pellet.Rehydrate DNA.

Discard supernatant.Add ethanol.

Centrifuge.

Centrifuge.

Discard supernatant.Vortex pellet. Add Nuclei Lysis Solution.Mix. Add ProteinPrecipitation Solution.

Centrifuge.

Add Cell LysisSolution. Incubate.

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 Kitas 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 containsamples that were rehydrated at room temperature for 20 minutes. Lanes 4, 6, 8 and 10 containsamples rehydrated at 4°C overnight.

Flow chart of genomic DNA purification using the Wizard Genomic DNAPurification Kit.

Product Size Cat.#Wizard® Genomic DNA Purification Kit 100 isolations x 300µl A1120

500 isolations x 300µl A1125 100 isolations x 10ml A1620

For Laboratory Use.

Wizard® Genomic DNA Purification Kit

5

Geno

mic

DNAEasy-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, 1.2cmmouse tail or 5 x 106 cultured cells in a single prep.

• Yield: Purify 20–30µg of gDNA per prep from 1.2cm mouse tailclips with an A260/A280 ratio of 1.7.

Amplification of genomic DNA isolated from various tissue sources using the Wizard SVGenomic DNA Purification System. One microliter of purified genomic DNA from the indicatedtissues 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 (–) wereperformed as positive and negative controls, respectively.

M – + Tail

Liver

Heart

Brain

Tail

Liver

Heart

Brain

3710

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A

Spin Vacuum

3643

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02_2

A

Add Wizard® SV Lysis Buffer.

Centrifuge.

Bind DNA.

Wash, removing solution by centrifugation or vacuum.

Transfer spin column to a 1.5ml microcentrifuge tube (not provided). Centrifuge.

Mouse tail clipping or tissue sample Proteinase K (Cat.# V3021)* digestion in Digestion Solution.

Genomic DNA Purification from Mouse Tail Clipping or Tissue Sample

Genomic DNA Purification from Tissue Culture Cells

Wash tissue culture cells with 1X PBS.

Incubate at 55°C overnight (16–18 hours).

Add Wizard® SV Lysis Buffer.

Transfer lystate to minicolumn.

Elute genomic DNA.

Vacuum Adapter (Cat.# A1331)*

Vac-Man® Laboratory

Vacuum Manifold (Cat.# A7231)*

Flowchart of genomic DNA purification using the Wizard SV Genomic DNAPurification System. *May be purchased separately.

Product Size Cat.#Wizard® SV Genomic DNA Purification System 50 preps A2360

250 preps A2361

Wizard® SV Genomic DNA Purification System

(a) The PCR process is covered by patents issued and applicable in certain countries*. Promega does not encourage orsupport 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, effectiveMarch 28, 2006, European Pat. Nos. 201,184 and 200,362 will expire.

6

M –C +C C4 C5 C6 C7 C8 D4 D5 D6 D7 D8 M E4 E5 E6 E7 E8

3712

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A

Mouse Tail Clippings

Water Only

1 2 3 4 5 6 7 8 9 1 1 1

A

B

C

D

E

F

G

H

No cross-contamination. Genomic DNA purified from mouse tail clipping samples or water samples werepurified in adjacent wells of a 96-well plate. PCR products were amplified from 1µl of purified sample from eachwell using primers for mouse IL-1β. Ten microliters of PCR product were run on a 1.5% agarose gel andvisualized by staining with ethidium bromide. Expected PCR product from mouse tail clippings for IL-1β isapproximately 1.2kb. No PCR product was detected from water samples.

Easy-to-use rapid 96-well isolation of gDNA from cellsor tissue.

The Wizard SV 96 Genomic DNA Purification System provides:• Performance: Purified DNA PCR ready.

• Time savings: 96-well processing using either manual orautomated purification protocols for Beckman Coulter Biomek®

instruments

• Flexibility: Purify genomic DNA from up to 20mg of tissue, 1.2cmof mouse tail or 5 × 106 cultured cells per well.

• Convenient: Purified genomic DNA from 96 mouse tails in 45–60minutes, and from cultured cells in 30 minutes after lysis. Nocentrifugation step required. PCR-ready gDNA can be isolatedfrom tissue or mouse tails directly from lysate.

Flowchart of 96-well genomic DNA purification using the Wizard SV 96 GenomicDNA Purification System.

Wizard® SV 96 Genomic DNA Purification System

Average Yield of Genomic DNA Purified from Various Sources Usingthe Wizard SV and SV 96 Genomic DNA Purification Systems.

Amount of AverageSource Starting Material DNA YieldMouse Tail Clipping 20mg 20µg

Mouse Liver 20mg 15µg

Mouse Heart 20mg 10µg

Mouse Brain 20mg 6µg

CHO Cells 1 × 106 cells 5µg

NIH3T3 Cells 1 × 106 cells 9µg

293 Cells 1 × 106 cells 8µg

Add sample lysate.

3637

MA

02_2

A

Bind DNA.

Wash.

Elute genomic DNA.

Highly pure genomic DNA.

Product Size Cat.#Wizard® SV 96 Genomic DNA Purification System 1 x 96 preps A2370

4 x 96 preps A2371

7

Geno

mic

DNAWalk-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 high-quality genomic DNA from well-to-well and plate-to-plate.

• Time savings: Standardized protocol provides reproducible resultsin 1.5 hours. Let us program your robot – downloadable methodsavailable at: www.promega.com/automethods

PCR analysis of genomic DNA isolated from 200µl whole blood samples. PCRproducts from 5µl of genomic DNA isolated using the MagneSil Blood Genomic, MaxYield System. Lane M, PCR markers (Cat.# G3161); (+) positive control apc; (–) noDNA control. Lanes 1–9 contain PCR amplification product from purified humangenomic DNA.

Deck layout for the MagneSil Blood Genomic Max Yield System on the Beckman CoulterBiomek® FX.

– 318

M + – 1 2 3 4 5 6 7 8 9bp

XX XX XX XX

XX XX XX

XX XX

XX SWAP XX XX XX XX

XX XX XX XX P16 P20

Heat Block

4152

MA

05_3

A

Cleanup Tips

P1

P2

P3

P4

P5

P6

P7

P10 Orbital1 P7

P8

P9

P11

P12

P13

P14

P15

P18

P19

WS1 Blood Sample Resin Disp Heat Block

2nd Tips Lysis Resin MagnaBot® Lysis Wash Lysis Disp Elution Solution

3rd Tips Wash Plate Salt Wash Salt Disp Elution Disp

4th Tips Elution Tips Alcohol Wash Alcohol Disp

Product Size Cat.#MagneSil® Blood Genomic, Max Yield System 1 x 96 preps MD1360 For Laboratory Use.

Purification of apoptotic DNA using the MagneSil Blood Genomic, Max YieldSystem. In some cases, blood samples that are stored or shipped at elevatedtemperatures contain white blood cells undergoing apoptosis. The MagneSil BloodGenomic System purifies DNA across the broad size range seen in a typical apoptoticladder. Genomic DNA was isolated from blood that had been stored at the indicatedtemperatures for 8 days.

4°C 21°C –20°CM 1 2 3 4 5 6 7 8 9

4235

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MagneSil® Blood Genomic, Max Yield System

8

Walk-away, high-throughput fixed-yield genomic DNAisolation 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 forgenotyping.

• Consistent results: Obtain 1µg (fixed yield) of highly pure DNAfrom 60µl of anti-coagulated blood.

• Time savings: Complete walk-away high-throughput 96-well takes1-hour.

Place the blood samples onto the instrument and come back in anhour to purified and normalized DNA ready for PCR! Consistent yield of genomic DNA purified with the MagneSil ONE, Fixed Yield

System. Genomic DNA standards were loaded onto the gel at the followingconcentrations: 250, 125, 63 and 31ng. Forty-eight blood samples were purified withthe MagneSil ONE System, and 10µl of the 200µl total elution volume for each samplewas loaded on the gel and visualized by ethidium bromide staining.

Deck layout for the MagneSil ONE Fixed Yield Blood Genomic System on the Beckman CoulterBiomek® FX.

MagneSil® ONE, Fixed Yield Blood Genomic System

ng control

25012

563 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

17 18 19 20 21 22 23 24 25 26 27 28

37 38 39 40 41 42 43 44 45 46 47 48

29 30 31 32 33 34 35 36

4231

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07_3

A

SC1 XX XX XX XX

SWAP P5 P9 P13

XX

XX

TR1 XX XX XX XX Magbead

XX XX XX XX XX Shaker1

4153

MA

05_3

A

Cleanup Tips

Orbital1 P4

P3

P2

P1

P8

P7

P6

P12

P11

P10

P16

P17

P15

P14

Resin Disp MagnaBot®

Working Plate

Wash Tips Wash Solution Alcohol Disp

Lysis Disp

Elution Solution Elution Disp

Elution Tips

Alcohol Disp WS1

Heat Block

Lysis Wash

Lysis Resin

Heat Block

Product Size Cat.#MagneSil® ONE, Fixed Yield Blood Genomic System 1 × 96 preps MD1370 For Laboratory Use.

9

Geno

mic

DNAEasy, 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-intensivesteps normally associated with plant genomic DNA preps. Manualprotocol takes about 45 minutes while the Beckman CoulterBiomek® FX robot will complete purification in about 25 minutes.

• Consistent DNA quality: From well-to-well, plate-to-plate across abroad variety of plant material.

• Ease of use: Fewer steps and non-clogging magnetic particletechnology.

• Adaptable chemistry: Scalable purification for standard or deepwell plates. Modified chemistry for fixed yield purification anddirect genotyping of gDNA.

Time Required to Isolate gDNA from Plant Tissue Using Three Listed ManualProcedures.

Method Steps Time

Wizard Magnetic 96 DNA Plant System 11 40 minutesCommercial membrane plate 17 55 minutesCTAB homebrew method 16 135 minutes

Examples of Plant Sample Types Successfully Processed Using the WizardMagnetic 96 DNA Plant System.Arabidopsis Cotton seed SoybeanCabbage seed Grass seed SquashCanola leaf Green pepper seed Squash seedCanola seed Lettuce Strawberry leaf*Carrot seed Milkweed leaf Sunflower seedChicory leaf Potato tuber Tobacco seedlingChives Radish leaf Tomato leafCorn leaf Rice leaf Tomato seedCotton leaf* Sorghum Watermelon seed* These samples need the addition of PVPP to the lysis buffer to remove phenolic compounds that inhibit PCR.

Place fresh leaf or seed samplein 96 deep-well plate in the presenceof Lysis Buffer A. Add 1 or 2grinding beads.

Transfer supernatant to a cleanplate. Add Lysis Buffer B/MagneSil® Paramagnetic Particle mixture and mix well.Incubate.

Place the plate on the MagnaBot®96 Magnetic Separation Device. Discard liquid.

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.

Grind. Centrifuge.

32

20

MA

01

_1

A

Flowchart of plant genomic DNA purification using the Wizard Magnetic 96 DNAPlant System.

Product Size Cat.#Wizard® Magnetic 96 DNA Plant System 2 x 96 preps FF3760

4 x 96 preps FF3761

Wizard® Magnetic 96 DNA Plant System

10

Analysis of DNA purified from paraffin-embedded, formalin-fixed 10µm thin sections using theMagneSil Genomic, Fixed Tissue System. Purified DNA was amplified and then analyzed on anABI PRISM® 3100 Genetic Analyzer. Panel A. Amplification with a set of 16 fluorescently labeledprimers. Amplification products range in size from 104 to 420 bases. Panel B. A 972-basefragment amplified using an amelogenin primer set. Panel C. A 1.8kb fragment amplified fromthe APC gene. Results will vary depending on the degree of cross-linking due to formalin fixation.

Flowchart of DNA isolation from formalin-fixed paraffin embedded tissue using theMagneSil Genomic, Fixed Tissue System.

MagneSil® Genomic, Fixed Tissue System

3931

TB

12_2

A

Size Markers APC1.8Kb

Fragment

926 baseSize

Marker

972 baseAmelogenin

Fragment

100 200 300 400 500600

A.

B.

C.

Add Incubation Buffer/ Proteinase K solution to tissue sample. Incubate overnight at 56°C.

Add Lysis Buffer and Resin. Vortex.

Place on Magnetic Standand discard solution.

Wash with Lysis Buffer.Wash with prepared 1X WashBuffer. Repeat twice for a total of three washes.

Air dry Resin.

Add Elution Buffer. Heat.

Remove tubes from heat, vortex,and place in Magnetic Stand.

Transfer DNA to container of choice.

3742

MA

06_2

A65°C

Product Size Cat.#MagneSil® Genomic, Fixed Tissue System 100 samples MD1490For Laboratory Use.

Easy-to-use isolation of high-quality DNA from fixed-tissue 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 aslarge as 1,800bp.

• Quick results: Proteinase K treated sample processed to purifiedDNA in under 30 minutes, 6–12 samples in under 1 hour.

• Consistent amplification: Ultraclean DNA free of inhibitors ofamplification including very small fragments of DNA.

11

Geno

mic

DNAWalk 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 andcuts labor costs.

• Consistent results: Well-to-well 2–6µg of high-quality DNA perprep from 200µl of anti-coagulated whole blood.

• Simple method: One instrument and one chemistry for gDNApurification, no training or modification of protocols necessary.

• Save time: Automated purification method takes 25 minutes tocomplete.

Yield gel of genomic DNA isolated from 200µl of liquid blood. Fifteen representativesamples (single instrument run) of DNA purified using the MagneSil KF GenomicSystem from blood. Ten microliters of the 200µl total elution volume were loaded onthe gel and visualized by ethidium bromide staining. Lanes 6 and 12 left intentionallyblank.

Multiplex amplification of Factor II (345bp) and Factor V (225bp). Genomic DNA purified from200µl human whole blood as recommended in Promega Technical Bulletin #TB322 MagneSil KF,Genomic System. Figure courtesy Dr. Greg Tsongalis, Hartford Hospital, USA.

4212

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

4213

TA

06_3

A

Product Size Cat.#MagneSil® KF, Genomic System 200 preps MD1460For Laboratory Use.

MagneSil® KF, Genomic System

12

Wizard® Magnetic DNA Purification System for Food

3019

TA

06_3

A

0.0Mbp

10,000 –

1 2 3 4 5 6 7 8 9 10

1,000 –

500 –300 –150 –

GMO Maize (%)0.1 0.5 2.0

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

195bp (S35)180bp (NOS)

bp M

1,000 –500 –300 –

150 –

– 35S

– NOS

– C – 35S

– NOS

– C – 35S

– NOS

– C – 35S

– NOS

– C

Cornmeal(GMO+)

Corn Seed(GMO–)

3016

TA

06_3

A

Prepare Lysate.

Centrifuge.

Bind DNA.

Capture MagneSil® PMPs.

Wash MagneSil® PMPs.Dry particles.

Add sterile water.Vortex and incubate.

Capture particles.

Elute DNA.

Food Sample

3013

MA

07_0

A

Duplicate Fluka maize reference standard maize samples containing up to 2% GMO maizewere processed with the Wizard Magnetic DNA Purification System for Food. Ten percent of100µl eluates of DNA in duplicate were loaded on a 1% agarose gel. The 0% standard shows agreater yield and higher molecular weight DNA because non-GMO maize undergoes minimalprocessing in the preparation of the reference standard. Lanes 1 and 2, 1kb DNA Step Ladder(Cat.# G6941) and PCR Markers (Cat.# G3161), respectively.

DNA isolated from cornmeal and ground corn seed using the Wizard Magnetic DNAPurification System for Food. Detection of GMO sequences by PCR was performed using 1µl ofDNA sample per template. The first three replicate sample sets (lanes 1–9) are from GMO+cornmeal. Samples represented in lanes 10–12 are from GMO– corn seed; nonspecific bands areseen in these 35S and NOS lanes. Lane M, PCR Markers (Cat.# G3161). Lanes labeled C:chloroplast PCR positive controls.

Flowchart of genomic DNA purification from food using the Wizard Magnetic DNAPurification 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 200mgto 5g food samples.

• Consistent results: Ultraclean gDNA of a broad size range free ofinhibitors of amplification.

Product Size Cat.#Wizard® Magnetic DNA Purification System for Food 200 preps FF3750

400 preps FF3751

13

Geno

mic

DNA

Product Size Cat.#Wizard® Genomic DNA Purification Kit* 100 isolations x 300µl A1120

500 isolations x 300µl A1125100 isolations x 10ml A1620

Cell Lysis Solution* 1 liter A7933Nuclei Lysis Solution* 50ml A7941

1 liter A7943Protein Precipitation Solution* 25ml A7951

350ml A7953DNA Rehydration Solution* 50ml A7963RNase A Solution, 4mg/ml* 1ml A7973Proteinase K* 100mg V3021

Product Size Cat.#Wizard® SV Genomic DNA Purification System(a) 50 preps A2360

250 preps A2361Wizard® SV 96 Genomic DNA Purification System(a) 1 x 96 preps A2370

4 x 96 preps A2371Proteinase K* 100mg V3021Wizard® SV Lysis Buffer* 50ml Z3052Wizard® SV Wash Solution* 185ml A1311Neclei Lysis Solution* 50ml A7941EDTA, 0.5M (pH 8.0),Molecular Grade* 100ml V4231RNase A Solution, 4mg/ml* 1ml A7973

Product Size Cat.#MagneSil® Blood Genomic,Max Yield System(b)* 1 x 96 preps MD1360Anti-Foam Reagent* 300µl MD1431MagneSil® Paramagnetic Particles* 25ml MD1441Salt Wash, Blood* 90ml MD1401Alcohol Wash, Blood* 70ml MD1411Elution Buffer, Blood* 45ml MD1421

Product Size Cat.#MagneSil® ONE, Fixed Yield Blood Genomic System(b)* 1 x 96 preps MD1370Lysis Buffer, Blood* 160ml MD1392Anti-Foam Reagent* 300µl MD1431MagneSil® PMPs—Fixed Yield* 25ml MD1451Alcohol Wash, Blood* 120ml MD1412Elution Buffer, Blood* 45ml MD1421

Product Size Cat.#Wizard® Magnetic 96 DNA Plant System(b) 2 x 96 preps FF3760

4 x 96 preps FF3761Wash Buffer, Plant 40ml A3811

Product Size Cat.#MagneSil® Genomic, Fixed Tissue System* 100 samples MD1490

Product Size Cat.#MagneSil® KF, Genomic System* 200 preps MD1460MagneSil® KF, Paramagnetic Particles* 40ml MD1471Lysis Buffer, KF* 160ml MD1521

Product Size Cat.#Wizard® Magnetic DNA Purification System for Food(b) 200 preps FF3750

400 preps FF3751RNase A Solution, 4mg/ml* 1ml A7973Lysis Buffer A, Food 100ml A8191Lysis Buffer B, Food 100ml Z3191Precipitation Solution, Food 150ml Z3201

Hard Ware Devices/AccessoriesProduct Size Cat.#Vac-Man® Laboratory Vacuum Manifold, 1 each A7231 20-sample capacity Vacuum Adapters 20 each A1331Vac-Man® 96 Vacuum Manifold 1 each A2291Deep Well MagnaBot® 96 MagneticSeparation Device* 1 each V3031MagnaBot® Spacer 1/8 inch 1 each V8581Collection Plates (4-pack) 1 each A9161Heat Transfer Block 1 each Z3271MagnaBot® 96 Magnetic Separation Device 1 each V8151MagnaBot® Spacer 1 each V8381MagnaBot® Adapter T1 1 each V8481MagneSphere® Technology Magnetic Separation Stand (two-position) 1.5ml Z5332MagneSphere® 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. 730718 and other patents and patents pending.(b)U.S. Pat. Nos. 6,027,945, 6,368,800 and 6,673,631, Australian Pat. No. 732756 and other patents and

patents pending.

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.

Ordering Information

14

Information...always at your finger tips!

Learn how…ask Promega

DNA Analysis NotebookGet 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 evenRNAi.

Call your local Promega representative to get your free copy or visit us on-lineat: www.promega.com/guides

#BR120

#BR129

#BR152

15

Plas

mid

DNA

Purify Plasmid DNA...

Purify Plasmid on Any Scale: • Miniprep

• Midiprep

• Maxiprep

• Megaprep

• Multiwell-plate formats

• Full automation

...at high yield, on any scale from manual to automatedplate 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 plasmidDNA purification systems. You choose the separation method bestsuited to your needs.

• Your choice of throughput: Both membrane-based and magnetic-basedplasmid purification systems are available in manual and automatedformats.

• Systems tailored to your purification scale: From miniprep tomegaprep scale, there is a plasmid purification system for every need.

• Fully automated systems for high-throughput users: Automatedmethods and personalized automation support are available for all highthroughput systems.

• Increased productivity: Save time and labor with our easy to useprotocols.


Culture Spin- Vacuum- Magnetic Manual Automated Volume Protocol Protocol Technology Handling Handling

PureYield™ Plasmid Midiprep System (Cat.# A2492, A2495) 50–250ml

Wizard® Plus SV Minipreps (Cat.# A1330, A1340, A1460, A1470) 1-10ml

Wizard® SV 96 Plasmid System (Cat.# A2250, A2255) multiwell

Wizard® MagneSil Tfx™ System (Cat.# A2380, A2381) multiwell

Wizard® Plus Mini and Midipreps Systems (Cat.# A7100, A7500, A7510, A7640) 1-100ml

Wizard® Plus Maxi and Megapreps Systems (Cat.# A7270, A7300) 100-1,000ml

Wizard® PureFection System (Cat.# A2150, A2160, A2170) 5-125ml

Wizard® MagneSil® Plasmid Purification System (Cat.# A1630, A1631) multiwell

A Comparison of Plasmid DNA Purification Systems.

16

Product Size Cat.#PureYield™ Plasmid Midiprep System 25 preps A2492

100 preps A2495For Research Use.

Fast and reliable membrane-based column method formidiprep 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 concentratedplasmid DNA gives proven performance in transfection in vitroexpression and other molecular biology applications.

• Spend less time purifying DNA: High yield gives you moreexperiments per midiprep.

• Flexibility: The high binding capacity of PureYield columns allowsplasmid purification from large culture volumes (250ml), providingmaxiprep yield from a midiprep system.

The PureYield™ Plasmid Midiprep System

Comparison of time required per midiprep using different systems. Each systemprotocol was performed according to the manufacturer’s instructions using 50ml of an overnightculture of JM109 bacteria transformed with a high copy plasmid (pGEM®-3 plasmid). Total timeto perform the midiprep is noted.

4587

MB

PureYield™ System Done in 30 Minutes

Wizard® System 99 Minutes

Qiagen Plasmid 200 Minutes

Proper assembly of Lysate Clearing Column (blue) and DNA Binding Column(white) for use with the PureYield Plasmid Midiprep System vacuum-methodpurification protocol.

4594

CA

4701MA

DNA Preparation Methods

Qiagen Eppendorf PureYield™System

01,0002,0003,0004,0005,0006,0007,0008,000

Fire

fly L

ucife

rase

(RLU

)

Comparison of transfection efficiency of purified plasmid DNA prepared using differentmidiprep systems. Comparison of transfection of plasmid DNA purified with the PureYieldSystem and other midiprep systems. psiCHECK-2™ Vector (Cat.# C8021), which carries a fireflyluciferase gene, was isolated from E.coli using either the PureYield System, the HiSpeed™ QiagenPlasmid Midi Kit or the Eppendorf Perfectprep® Plasmid Midi Kit. HeLa cells were transfectedusing 0.07µg of DNA in a total of 25µl. The firefly luciferase signal was monitored with the Dual-GloTM Luciferase Reporter System (Cat.# E1910).

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Wizard® Plus SV Minipreps DNA Purification System

Low-throughput, silica-membrane column-basedminiprep 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 influorescent sequencing reactions to ensure read lengths of at least500 base pairs with 98% accuracy.

• Time savings: Isolate 20 minipreps in less than 45 minutes.

• Compatibility with EndA+ strains: Incorporation of an alkalineprotease step allows DNA isolation from problematic strains.

Product Size Cat.#Wizard® Plus SV Minipreps DNA Purification System 50 preps A1330Wizard® Plus SV Minipreps DNA Purification System + Vacuum Adapters 50 preps A1340Wizard® Plus SV Minipreps DNA Purification System 250 preps A1460Wizard® Plus SV Minipreps DNA Purification System + Vacuum Adapters 250 preps A1470For Laboratory Use.(a) U.S. Pat. No. 4,766,072.(b) U.S. Pat. No. 5,955,363.

pGEM® 3-Zf(+) plasmid sequenced with T7 sequencing primer using DNApurified by the Wizard Plus SV Minipreps DNA Purification System.

– 10.0– 6.0– 4.0

– 2.0– 1.5

– 1.0– 0.75– 0.50– 0.25

– 3.0

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High-quality restriction digests using plasmid purified with the Wizard Plus SVMinipreps DNA Purification System. High-copy pGEM®-3Zf(+) Vector(a) (lanes 1–3) andlow-copy pALTER®-1 Vector(a,b) (lanes 4–6) were each digested in two separate Promegarestriction enzyme reactions. All lanes show reproducible and efficient cutting of the plasmid DNAand are measured against the 1kb DNA Ladder (Cat.# G5711).

18

Wizard® SV 96 Plasmid DNA Purification Systems

High-throughput 96-well silica-membrane plate basedplasmid DNA purification in automated or manualformat

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 with98% accuracy.

• High quality at high speed: Process 96-wells of bacterial culturein 60 minutes or less, using a 96-well, vacuum filtration step tosimultaneously clear bacterial cell lysates and bind DNA—nocentrifugation required!

• A walk-away, automated procedure: No manual interventionrequired once the E.coli cell pellets are placed on the robot deck.

• Hassle-free automated methods: Automated methods andsupport information available at: www.promega.com/automethods/

• Ease-of-use and handling: The novel plate design prevents crosscontamination during sample processing. Filtrate waste productsare delivered directly to a vacuum trap, eliminating the need toempty waste collection vessels during the purification procedure.

Product Size Cat.#Wizard® SV 96 Plasmid DNA Purification System 1 x 96 preps A2250

5 x 96 preps A2255Wizard® SV 9600 Plasmid DNA Purification System* 100 x 96 preps A2258Cat.# 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.

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The Wizard® SV 96 System operates as a true “walkaway” system on automated purificationplatforms such as the Beckman Coulter Biomek® FX laboratory automation workstation. It canalso be used in a manual format.

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Lysate Clearing Plate

Binding Plate

Elution Plate

Overnight cultures

Resuspend pelleted bacterial cells.Lyse.Neutralize.

Transfer Lysates.

Clear lysates.

Bind DNA.

Wash.

Elute DNA.

Highly pure plasmid DNA.

Wizard SV 96 Plasmid DNA Purification System protocol overview. Automatedprotocols are available at: www.promega.com/tbs

Wizard SV 96 is tested to ensure that purifiedDNA meets or exceeds 600 bases with 98%accuracy in fluorescent sequencing analysis.

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Wizard MagneSil Tfx™ System

Transfection-quality plasmid DNA purification in ahigh-throughput 96-well miniprep format

The Wizard MagneSil Tfx System provides:• Lowered endotoxin plasmid DNA: Use of the endotoxin removal

resin cuts endotoxin carryover as much as 95% over automatedstandard sequencing grade plasmid systems. Lower endotoxinlevels produce better transfection efficiency.

• Improved mammalian protein expression: Three- to five-foldincrease in protein expression compared to plasmid isolated froman automated sequencing-grade plasmid purification system.

• High-quality at high speed: Process 96-wells of bacterial culturein under 45 minutes with a Beckman Coulter Biomek® FX, using aparamagnetic particle-based purification method for lysate clearingand DNA capture.

• Hassle-free automated methods: Automated method and supportinformation available at: www.promega.com/automethods/

Flow chart of Transfection-grade plasmid DNA purification using the WizardMagneSil Tfx System.

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Wash

Bind DNA

Elute

Wash

Dry

Bind DNA

Remove Endotoxin

Clear Cell Debris

Sample-to-sample yield of plasmid DNA purified using the Wizard MagneSil Tfx System. Deep-well 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 protocolprovided in the Wizard MagneSil Tfx™ System Technical Bulletin #TB314. Each bar represents theaverage of 8 samples. Samples were quantitated by gel electrophoresis.

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Yields of Plasmid DNA20151050

(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 theUniversity of California for all other fields) is needed for any commercial sale of nucleic acid contined within orderived from this product.

Product Size Cat.#Wizard MagneSil Tfx™ System 4 x 96 preps A2380

8 x 96 preps A2381

20

250

200

150

100

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25

Culture Volume (ml)

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High Copy NumberLow Copy Number

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Plasmid DNA yield obtained using the Wizard Plus Midipreps DNA PurificationSystem. 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 alow-copy plasmid, pBR322, prepared in E. coli strain DH5α™.

Mini-, Midi-, Maxi- and Megaprep scale plasmid DNApurification 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 as15 minutes, Midipreps in 90 minutes, and Maxi- and Megaprepsin 3 hours.

• Sequencing-quality DNA: Each Wizard Plus System is qualitytested in fluorescent sequencing reactions to ensure read lengthsof at least 500 base pairs with 98% accuracy.

• Safety and convenience: No phenol extraction or precipitationrequired.

• High yields: Using a high copy number plasmid, the followingyields are obtained: 1–10µg DNA from 1–3ml culture; up to 200µgDNA from 10–100ml culture; up to 300µg–1mg from 100–500mlculture; and >1mg from 1,000ml culture.

Wizard® Plus DNA Purification Systems

DNA purification using the Wizard PlusSystems requires use of a vacuum sourceand either a lab-assembled or commercial

manifold such as Promega’s Vac-Man®

Laboratory Vacuum Manifold (20-samplecapacity).

Product Size Cat.#Wizard® Plus Minipreps DNA Purification System 50 preps A7100

100 preps A7500250 preps A7510

Wizard® Plus Midipreps DNA Purification System 25 preps A7640Wizard® Plus Maxipreps DNA Purification System 10 preps A7270Wizard® Plus Megapreps DNA Purification System 5 preps A7300

Fluorescent dye primer sequencing of pGEM® Vector purified using the Wizard PlusMinipreps DNA Purification System. The system is tested to ensure that purifiedplasmid DNA can be sequenced at ≥98% accuracy over 500 bases using dye primerchemistry and an ABI PRISM® 377 DNA sequencer.

(a)U.S. Pat. No. 4,766,072.

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Low-throughput, transfection-grade plasmid DNApurification 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 standardsequencing grade plasmid systems. Lower endotoxin levels producebetter transfection efficiency.

• Safety and convenience: No phenol extraction or precipitationrequired, using a magnetic-based purification method for lysateclearing and DNA capture.

• High yield: 40µg DNA from 5ml culture; scale-up capacity up to~1mg.

• Fast, efficient purification: The hand-held MagneSil MagneticSeparation Unit holds a 50ml tube and allows rapid and efficientcapture of magnetic particles and their attached DNA.

• Scalable format: Rapid purification from a range of starting volumesfrom midiprep (5–25ml) to maxiprep (26–125ml) scale.

Product Size Cat.#Wizard® PureFection PlasmidDNA Purification System 2 maxipreps/10 midipreps A2150

5 maxipreps/25 midipreps A216025 maxipreps/125 midipreps A2170

For Laboratory Use.

Alkaline LysisProduce cleared lysate.

Selective Removal of Endotoxin with MagnetEndotoxin Removal Resin clears bacterial lysate of endotoxin.

Bind Plasmid DNASelective binding of plasmid DNA using MagneSil® Paramagnetic Particles.

Wash & EluteWash particles to remove salts and elute in water. Ethanol precipitate and resuspend DNA at desired concentration.

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Overview of Wizard PureFection Plasmid DNA Purification System protocol.

0

100

200

300

400

500

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700

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Endo

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Endotoxin Removal Resin (mg) 23

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Removal of endotoxin from bacterial cell lysates using Endotoxin Removal Resin. Over 95% ofendotoxin is removed from bacterial lysates following a 5-minute incubation.

Wizard® PureFection Plasmid DNA Purification System

22

High-throughput 96-well plasmid DNA purification inan 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 influorescent sequencing reactions to ensure read lengths of at least600 base pairs with 98% accuracy.

• High-throughput: Process multiple plates without userintervention.

• A simple, fast method: Plasmid DNA isolation is accomplishedeasily, with a simple, magnetic-based procedure, using aparamagnetic particle-based purification method for lysate clearingand DNA capture.


Wizard® MagneSil® Plasmid Purification System

Accuracy by read length for plasmid DNA sequenced following isolation byMagneSil Paramagnetic Particles. Results demonstrate greater than 700 consecutivebases analyzed with greater than 98% accuracy of base identity.

500 600 70090

92

94

96

98

100

Accu

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(%)

Bases

2 O.D.4 O.D.6 O.D.8 O.D.

10 O.D.12 O.D.

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The Wizard MagneSil Plasmid Purification System is tested to ensure reliable DNArecovery. Plasmid DNA was purified from multiple samples grown to the statedO.D.600. DNA yield was measured on an agarose gel.

Product Size Cat.#Wizard® MagneSil® Plasmid Purification System 4 x 96 preps A1630

8 x 96 preps A1631100 x 96 preps A1635

Capture MagneSil® BLUEon MagnaBot® Device.

Capture the MagneSil® RED on the MagnaBot® Device.

Remove supernatant.

Clear Cell Debris

Bind DNA

Wash

Dry

Elute

Wizard MagneSil Plasmid Purification System protocol. Automated protocols are available at:www.promega.com/tbs

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MagneSil Paramagnetic Particles can berelied upon to purify high-quality template

for fluorescent sequencing.

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Product Size Cat.#PureYield™ Plasmid Midiprep System(a) 25 preps A2492

100 preps A2495

Product Size Cat.#Wizard® Plus SV Minipreps DNAPurification System(b,c)* 50 preps A1330Wizard® Plus SV Minipreps DNA Purification System + Vacuum Adapters(b,c)* 50 preps A1340Wizard® Plus SV Minipreps DNAPurification System(b,c)* 250 preps A1460Wizard® Plus SV Minipreps DNAPurification System + Vacuum Adapters(b,c)* 250 preps A1470Alkaline Protease Solution* 3ml A1441

Product Size Cat.#Wizard® SV 96 Plasmid DNAPurification System(b)* 1 x 96 preps A2250

5 x 96 preps A2255Wizard® SV 9600 Plasmid DNAPurification System 100 x 96 preps A2258Wizard® SV Wash Solution* 185ml A1311Wizard® SV 96 Wash Solution 370ml A1318Wizard® SV 96 Neutralization Solution* 500ml A1481

950ml A1488Wizard® SV 96 Cell Resuspension Solution* 500ml A7113

800ml A7118Wizard® SV 96 Cell Lysis Solution* 500ml A7123

800ml A7128Nuclease-Free Water* 150ml P1195Alkaline Protease Solution* 3ml A1441

Product Size Cat.#Wizard MagneSil Tfx™ System(a,d,e) 4 x 96 preps A2380

8 x 96 preps A2381Endotoxin Removal Resin 100ml A21914/40 Wash Solution 115ml A2221

Product Size Cat.#Wizard® Plus Minipreps DNAPurification System(f)* 50 preps A7100

100 preps A7500250 preps A7510

Wizard® Plus Midipreps DNAPurification System(f)* 25 preps A7640

Product Size Cat.#Wizard® Plus Maxipreps DNAPurification System(f)* 10 preps A7270

Wizard® Plus Megapreps DNAPurification System(f)* 5 preps A7300

Product Size Cat.#Wizard® PureFection Plasmid DNAPurification System(a,d)* 2 maxipreps/

10 midipreps A21505 maxipreps/25 midipreps A2160

25 maxipreps/125 midipreps A2170

Product Size Cat.#Wizard® MangeSil® PlasmidPurification System(d,e) 4 x 96 preps A1630

8 x 96 preps A1631100 x 96 preps A1635

MagneSil® RED 100ml A1641MagneSil® BLUE 100ml A2201Cell Resuspension Solution 500ml A7114Cell Lysis Solution 500ml A7124Neutralization Solution 500ml A7132Elution Buffer 500ml A1655

Hard Ware Devices/Accessories

Product Size Cat.#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 A1331Vac-man 96 Vacuum Manifold 1 each A2291Collar for Vac-Man 96 Vacuum Manifold 1 each A2311Wizard® SV 96 Binding Plates* 10 pack A2271

100 each A2278Wizard® SV 96 Lysis Clearing Plates 10 pack A2241

100 each A2248MagnaBot® 96 Magnetic Separation Device 1 each V8151Collection Plates (4-pack) 1 each A9161*For Laboratory Use.

(a)U.S. Pat. No. 6,194,562, Australian Pat. No. 740145, Canadian Pat. No. 2,329,067 and other patentspending.

(b)U.S. Pat. No. 5,981,235, Australian Pat. No. 729932 and other patents pending. (c)Australian Pat. No. 730718 and other patents and patents pending.(d)U.S. Pat. Nos. 6,027,945, 6,368,800 and 6,673,631, Australian Pat. No. 732756 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. 689815 and other patents and patents

pending.

MagneSil, pALTER, pGEM, Vac-Man and Wizard are registered trademarks of Promega Corporation.

24

www.promega.com

Scientists helping scientists to be successful!Get the best-in-class help and support you need from Promega.All Promega Technical Services Scientists have extensivelaboratory experience. All have life science degrees and mosthave post-graduate or post-doc lab experience. If you have aquestion about one of our products, or even about a productthat is not ours, feel free to contact us.

Visit our web site for 24-hour a day support. Find online protocols, productFAQs, application information, references, publications, calculators and manyother valuable support tools. It’s all easy to access and easy to understand.

■ Telephone■ E-mail■ Troubleshooting

experiments■ Automation installation

and support■ Global availability■ Multi-language capabilities

Telephone: 608-274-4330

Toll Free in USA: 800-356-5926

www.promega.com/techserv/

Superior Technical Services

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Purify DNA Fragments...

A Comparison of Fragment DNA Purification Systems.

...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 membranefiltration-based and magnetic particle-based systems. You choose theseparation method best suited to your needs.

• Your choice of throughput: Both membrane-based and magnetic-basedpurification systems are available in manual and automated formats.

• Simplicity and sophistication: From manual, single-tube purification tofull automation, there is a fragment purification system for every need.

• Fully automated systems for high-throughput users: Automatedmethods and personalized automation support are available for all highthroughput systems.

• Increased productivity: Save time and labor from easy to useprotocols.

• Experience you can trust: Have confidence in a supplier with over 25years 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

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)

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Flow chart of DNA fragment gel purification or direct PCR product purification using the Wizard SV Gel and PCR Clean-Up System.

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CentrifugeVacuum

Bind DNA

Wash, removing solution by centrifugation or vacuum

Transfer spin column to a 1.5ml microcentrifuge tube (not provided) Centrifuge

Excise gel slice and add equal volume of MembraneBinding Solution

Gel Purification

Direct PCR ProductPurification

Add equal volumeof Membrane BindingSolution to PCR reaction

50–65°C for 10minutes to solubilizegel slice

Transfer sample to SV Minicolumn

Elute DNA fragmentor PCR product

Vacuum Adapter

(Cat.# A1331)

Vac-Man® Laboratory

Vacuum Manifold (Cat.# A7231)

Wizard SV Gel and PCR Clean-Up System: Percent recovery of varioussized fragments directly from PCR.

DNA Fragment Size Percent Recovery

200bp 87%500bp 91%

1,000bp 92%1,500bp 92%3,199bp 87%

Product Size Cat.#Wizard® SV Gel and PCR Clean-Up System 50 preps A9281

250 preps A9282For Laboratory Use.

U

U: Unpurified P: Purified

100bp 500bp 1,000bp 3,199bp

M

3,000bp –

1,000bp –

P U P U P U P

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Gel analysis of DNA fragments before and after gel extraction using the Wizard SVGel and PCR Clean-Up System. DNA fragments of the sizes indicated were analyzedbefore (U; unpurified) and after (P; Purified) purification. The marker (M) used is the1kb DNA Ladder (Cat.# G5711).

Wizard SV Gel and PCR Clean-Up System: Percent recovery of varioussized fragments from agarose gels.

DNA Fragment Size Percent Recovery

70bp 39%85bp 55%

100bp 84%500bp 89%

1,000bp 92%3,199bp 95%9,416bp 95%

High recovery of concentrated DNA fragments over abroad 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 orTBE 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 agarosegels.

• An application-tested system: Tested to ensure performance ofpurified PCR products in fluorescent sequencing. Purified DNAroutinely achieves 700bp with >98% accuracy.


Wizard® SV Gel and PCR Clean-Up System

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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 1,000bpfragments is >90%, unaffected by common PCR additives.

• Gel slice purification: The system can be used to purify DNA fromgel slices, as well as directly from PCR.


• An application-tested system: Tested to ensure performance ofpurified PCR products in fluorescent sequencing and microarrayapplications.


Wizard® SV 96 PCR Clean-Up System

Product Size Cat.#Wizard® SV 96 PCR Clean-Up System 1 x 96 preps A9340

4 x 96 preps A93418 x 96 preps A9342

100 x 96 preps A9345For Laboratory Use.

A.

B.

100bp 200bp 300bp 500bp 1,000bp

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PM U P U P U P U P U

100 200 300 500 1,0000

102030405060708090

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ent R

ecov

ery

Size (bp)

Purification and recovery of PCR products. PCR fragments of 100, 200, 300, 500, and1,000 base pairs were purified using the Wizard SV 96 PCR Clean-Up System on theBeckman Coulter Biomek® 2000 robotic workstation. Panel A. Purified (P) andunpurified (U) fragments were separated on an ethidium bromide-stained, 2% agarosegel. Panel B. Percent recovery of purified PCR products was quantitated using aHitachi FMBIO® Fluorescent Scanner. Results show the mean and standard deviationfor 6 purified fragments of each size. For small PCR fragments (<500bp), optimalrecovery requires a 95% ethanol wash. For larger fragments (>500bp), optimal resultsare achieved using an 80% ethanol wash.

Wizard SV 96 PCR Clean-Up System protocol overview.

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Binding Plate

Collection Plate

Mix PCR sample and Membrane Binding Solution in equal volumes. Apply to Binding Plate.

Apply vacuum to bind PCR product to membrane.

Wash.

Elute bound PCR product with Nuclease-Free Water.

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Reliable low-throughput PCR and agarose gel sliceDNA 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 PCRproducts (300bp–1.5kb) directly from reactions.

• Efficiency: Process multiple samples simultaneously using theVac-Man Laboratory Vacuum Manifold (20 sample capacity).


• Confidence: Removal of excess nucleotides, primers and smallnonspecific amplification products such as primer-dimers fromPCR.

Wizard® PCR Preps DNA Purification System

1,000 –

bp

M M

500 –200 –50 –––––

Before Purification After Purification

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Recovery of PCR products using the Wizard PCR Preps Resin. A representative sample from thesimultaneous purification of 96 PCR reactions was chosen to determine the effectiveness of theDNA purification process. Equivalent amounts from before and after purification were separated ona 1% agarose gel and stained with ethidium bromide.

Product Size Cat.#Wizard® PCR Preps DNA Purification System 50 preps A7170

250 preps A2180For Laboratory Use.

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Purification and analysis of PCR products from low-melting agarose using theWizard 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.

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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 restrictionenzymes, phosphatases, kinases, DNA polymerases, exonucleasesand endonucleases (including DNase I but not RNases),mononucleotides and salts.

• Efficiency: Process multiple samples simultaneously using theVac-Man Laboratory Vacuum Manifold (20–sample capacity).


• 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

Product Size Cat.#Wizard® DNA Clean-Up System 100 preps A7280For Laboratory Use.

Removal of Common Impurities by the Wizard DNA Clean-Up System.

Contaminant Removed

Restriction enzymes YesHeat-stable restriction enzymes YesPhosphatases YesKinases YesLigases YesDNA polymerases YesTaq DNA polymerases YesExonucleases YesDNase I YesRNases NoMononucleotides YesSalts YesOligonucleotides No

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High-throughput automated 96-well or 384-wellsequencing reaction clean-up made simple andeconomical

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, witha simple, magnetic-based procedure.

• High quality purified DNA: Typical Phred 20 values of 600–700bases. 98% accuracy to at least 600 bases.

• Efficiency: Save labor with a walk-away high-throughputsequencing reaction clean-up automated method.


Wizard® MagneSil® Sequencing Reaction Clean-Up System

Product Size Cat.#

Sequencing Reaction Clean-Up in 96-Well PlatesWizard® MagneSil® Sequencing Reaction Clean-Up System 1 x 96 preps A1830

4 x 96 preps A18318 x 96 preps A1832

100 x 96 preps A1835

Sequencing Reaction Clean-Up in 384-Well PlatesMagneSil® GREEN 100ml A8231MagnaBot® 384 Magnetic Separation Device 1 each V8241384-Well Plate, Conical 10/pack V5311Cat.# A1830, A1831, A1832, A1835, A8231, V8241 For Laboratory Use.

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Clean-up is performed using the MagnaBot II Magnetic Separation Device (Cat.#V8351) accompanied by the Plate Clamp 96 (Cat.# V8251). These devices aredesigned to work with most robotic platforms.

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MagnaBot 384 Magnetic Separation Device (Cat.# V8241). The MagnaBot 384 isconstructed such that the magnetic field draws particles to the corners of theprocessing plate.

Phred 20 values for sequencing data from each well of a 96-well plate using the WizardMagneSil Sequencing Reaction Clean-Up System. Phred is a base-calling program that assignsan error probability to each base. A phred value of 20 for a given base assigns an error probabilityof 1 in a 100 for that base.

725–7

00

699–6

75

674–6

50

649–6

25

624–6

000

5

10

15

20

25

30

35

Phred 20 Read Length (bp)

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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.

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SequencingReaction

AddMagneSil® GREEN

Place onMagnaBot® II

RemoveSupernatant

Add Ethanol Wash Solution


RemoveSupernatant

Add Sequencing Gel Loading Solution


Transfer eluateto 96-well plate forsequence analysis

Binding Washing Elution 3169

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Overview of the Wizard MagneSil Sequencing Reaction Clean-Up System protocol.

A. MagneSil® PMPs

B. EtOH Precipitation

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A comparison of electropherograms of purified sequencing reactions analyzed on an ABI PRISM® 3700 DNA Sequencer. Panel A: Reactions purified withthe Wizard MagneSil Sequencing Reaction Clean-Up System. Panel B: Reactions purified by ethanol precipitation.

32

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 purificationmethod for DNA capture that is ideally suited for automation.

• A simple, fast method: No centrifugation or vacuum manifoldrequired.

• High quality DNA: Primer removal is consistently >98% withpurification of PCR fragments from 150bp–23kb in size.

• An application-tested system: Tested to ensure performance ofpurified PCR products in fluorescent sequencing and microarrayapplications.


Wizard® MagneSil® PCR Clean-Up System

3803

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200

300

500

600

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1,000

1,500

0

20

40

60

80

100

% R

ecov

ery

PCR Product Size (bp)

Recovery of PCR products of varying size using the Wizard MagneSil PCR Clean-Up System. Arange of sizes of PCR products, generated using PCR Master Mix(a) (Cat.# M7505), were recoveredusing the Wizard MagneSil PCR Clean-Up System on a liquid-handler. The yield of each PCRproduct 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 areshown.

MagnaBot 96 Magnetic Separation Device (Cat.# V8151).

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MagnaBot 384 Magnetic Separation Device (Cat.# V8241). The MagnaBot 384 isconstructed such that magnetic field draws the particles to the corners of the wells ofthe processing plate.

PCR Clean-Up in 96-Well Plates

Product Size Cat.#Wizard® MagneSil®PCR Clean-Up System(a) 4 x 96 preps A1930

8 x 96 preps A1931100 x 96 preps A1935

PCR Clean-Up in 384-Well Plates

Product Size Cat.#MagneSil® YELLOW 100ml A9231Wash Solution 500ml A8241MagnaBot® 384 Magnetic Separation Device 1 each V8241384-Well Plate, Flat 10/pack V5291Cat.# A1930, A1931, A1935, A8241, A9231, V8241 For Laboratory Use.

39

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(a) The PCR process is covered by patents issued and applicable in certain countries*. Promega does not encourage orsupport 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, effectiveMarch 28, 2006, European Pat. Nos. 201,184 and 200,362 will expire.

33

Frag

men

t DNA

PCRReactions

AddMagneSil® YELLOW

Place onMagnaBot® 96Device

RemoveSupernatant

DNA Binding

AddWash Solution

Place onMagnaBot® 96 Device

RemoveSupernatant

Washing

AddNuclease-FreeWater

Place onMagnaBot® 96Device

Transfer eluateto 96-well plate.

Elution of DNA 3288

MB

03_1

A

Repeat washcycle 2X with80% ethanol.

Overview of the Wizard MagneSil PCR Clean-Up System protocol.

Unpurified

PurifiedM

bp

500 –

1,500 –

100 –

100%

50%

25%

12.5%

6.25%

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The Wizard MagneSil PCR Clean-Up System removes primer-dimers from PCR products withhigh 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 visiblein these lanes. Eight replicates purified using the Wizard MagneSil PCR Clean-Up System show the506bp 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. LaneM, 100bp DNA Ladder (Cat.# G2101).

34


Product Size Cat.#Wizard® SV Gel and PCR Clean-Up System* 50 preps A9281

250 preps A9282Wizard® SV 96 PCR Clean-Up System(a)* 1 x 96 preps A9340

4 x 96 preps A93418 x 96 preps A9342

100 x 96 preps A9345Membrane Binding Solution* 20ml A9301

Product Size Cat.#Wizard® PCR Preps DNA Purification System(b)* 50 preps A7170

250 preps A2180

Product Size Cat.#Wizard® DNA Clean-Up System(c)* 100 preps A7280

Product Size Cat.#Wizard® MagneSil® Sequencing Reaction Clean-Up System(d)* 1 x 96 preps A1830

4 x 96 preps A18318 x 96 preps A1832

100 x 96 preps A1835MagneSil® GREEN* 100ml A8231

Product Size Cat.#Wizard® MagneSil® PCR Clean-Up System(d)* 4 x 96 preps A1930

8 x 96 preps A1931100 x 96 preps A1935

MagneSil® YELLOW* 100ml A9231Wash Solution* 500ml A8241Nuclease-Free Water* 150ml P1195


Product Size Cat.#Vac-Man® 96 Vacuum Manifold 1 each A2291Vacuum Adapters 20 each A1331Wizard® SV 96 Binding Plates* 10 pack A2271

100 each A2278MagnaBot® II Magnetic Separation Device 1 each V8351Plate Clamp 96 1 each V8251Plate Stand 1 each V8261MagnaBot® 96 Magnetic Separation Device 1 each V8151Plate Clamp 96 1 each V8251Plate Stand 1 each V8261MagnaBot® Spacer 1 each V8381MagnaBot® Adapter T1 1 each V8481Collection Plates (4-pack) 1 each A9161MagnaBot® 384 Magnetic Separation Device* 1 each V8241384-Well Plate, Conical 10 pack V5311384-Well Plate, Flat 10 pack V5291*For Laboratory Use.

(a)Australian Pat. No. 730718 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. 689815 and other patents and patents

pending.(d)U.S. Pat. Nos. 6,027,945 and 6,368,800, Australian Pat. No. 732756 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.

35

Tota

l RNA

Isolate RNA...

...from a variety of sample materials, on a scale of yourchoosing.

Promega’s Total RNA and mRNA Isolation Systems provide:

• Your choice of purification method: Promega provides a range ofsolution, membrane filtration-based and magnetic particle-basedsystems. You choose the separation method best suited to your needs.

• Your choice of throughput: Both membrane-based and magnetic-basedRNA isolation systems are available in manual and automated formats.

• Systems tailored to your purification scale: Mini-isolation from smallsample sizes to scaleable, solutions-based formats, there is a total RNApurification system for every need.

• Direct isolation of total RNA or mRNA from biological materials:Systems are available for direct isolation from a variety of startingmaterials.

• Increased productivity: Save time and labor with our easy to useprotocols.


Total Animal Culture Plant Manual Automated Magnetic Spin VacuumRNA Blood Bacteria Tissue Cells Material Handling Handling Technology Protocol Protocol

Total RNA Isolation Systems

RNAgents® Total RNA Isolation System (Cat.# Z5110)

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)

Purify RNA From:• Blood

• Tissue

• Cultured Cells

• Plant Material

• Bacteria

A Comparison of RNA Isolation Systems.

36

Scalable solution-based total RNA purification formaximum flexibility

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 oftissue, or from 105–108 cultured cells.

• Simplicity: A solution-based protocol for RNA isolation that canbe 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, plantleaf, etc.

• Maiximum flexibility: Denaturing Solution available separately formaximum flexibility, allowing you the option to supply the othersystem components (phenol/chloroform, isoamyl alcohol andisopropanol).

RNAgents® Total RNA Isolation System

Product Size Cat.#RNAgents® Total RNA Isolation System Scalable Z5110For Laboratory Use.

Average Yields of Total RNA from a Variety of Tissues.

Tissue RNAgents Total RNA Yield

HeLa cells 1.6mg RNA/108 cellsHuman white blood cells 1.3mg RNA/108 cells Mouse intestine 2.3mg RNA/g tissueMouse spleen 8.3mg RNA/g tissueMouse lung 1.9mg RNA/g tissueMouse kidney 3.1mg RNA/g tissueMouse liver 6.6mg RNA/g tissue

1 2 3 4 5 6

– 28S

– 18S

0349

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Analysis of RNA isolated from various tissues with the RNAgents System. RNA was isolatedfrom 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 eachsample was resolved on a 1% denaturing agarose gel (2.2M formaldehyde).

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E. coli Control Array

Cy®3 Cy®5

Cy®3: β-actin Cy®5: Kan

B. Jurkat Control Array

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®3target and another with a Cy®5 target. Panel B: Hybridization results for the JurkatControl Array. Fluorescent Cy®3 and Cy®5 targets were co-hybridized to the samearray, demonstrating specificity for their complementary sequences.

37

Tota

l RNA

Simple and reliable total RNA purification fromtissues, 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 phenolextraction required.

• Confidence in your results: Includes a DNase I treatment step toensure removal of genomic DNA. No detectable DNase I carryoverto the final RNA preparation.

• Peace of mind: Only system that includes DNase treatment step inthe standard protocol.

• Applications tested RNA: RNA suitable for all routine molecularbiology applications, including RT-PCR and Northern blotting.

SV Total RNA Isolation System

The vacuum protocol for the SV Total RNAIsolation System requires use of the

Vacuum Adapters and a vacuum manifoldsuch as Promega’s Vac-Man® LaboratoryVacuum Manifold (20-sample capacity).

Product Size Cat.#SV Total RNA Isolation System 10 preps Z3101

50 preps Z3100250 preps Z3105

For Laboratory Use.

bp1,500 –

1,000 –700 –

400 –

M 5µl 5µl 10µl 15µlβ-actin APC

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 primerscomplementary to human β-actin or human Adenomatous Polyposis Coli (APC) genewith the Access RT-PCR System(a,b) (Cat.# A1280, A1250).Average Yields of Total RNA Using SV Total RNA Isolation System.

Maximum Avg. Yield Avg. YieldAmount to per Prep per mg

Samples Process (µg) Tissue (µg) A260/A280

Mouse TissuesLiver 30mg 131 4.4 1.9Kidney 20mg 44 2.2 1.9Spleen 15mg 79 5.3 1.9Brain 60mg 39 0.65 2.1Muscle 30mg 22 0.73 2.1

Rat TissuesPancreas 30mg 100 3.5 1.9Heart 60mg 16 0.27 2.1Lung 60mg 36 0.6 2.1

BacteriaE. coli 1 x 109 cells 36 N/A 2.0

YeastS. cerevisiae 4 x 107 cells 19 N/A 2.1

PlantTomato Leaf 30mg 4.6 0.15 2.0

Cell LineRAW264.7 5 x 106 cells 51 N/A 2.1

N/A = Not Applicable.

Average yield is that of RNA only. Other commercial systems may appear to give higher yields, but this isfrequently due to DNA carryover.

38

Homogenize samples (tissues, cultured cells or white blood cells) in SV RNA Lysis Buffer.

Transfer to fresh tube; add 95% Ethanol and mix.

Transfer to Spin Column Assembly.

Elute RNA into Elution Tube.

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.

Spin

Remove lysate by vacuum.

Wash with SV RNA Wash Solution.

DNase Treatment. Add SV DNase Stop Solution.

Wash 2X with SV RNA Wash Solution.

Vacuum

Total time: 60-70 minutes

Remove Spin Basket and place in collection tube. Centrifuge. Place Spin Basket in Elution Tube.

Transfer to fresh tube and add SV RNA Dilution Buffer; mix and centrifuge.

1

Promega

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

1906

TA

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Competitor

Formaldehyde gel electrophoresis of RNA. RNA was isolated from 30mg of livertissue following the SV Total RNA Isolation System protocol (lanes 1–6) and acompetitors protocol (lanes 7–12). Ten percent (10µl) of the final prep volume of100µl for each sample was analyzed by electrophoresis in a 1% MOPS-EDTA-formaldehyde agarose gel and visualized by including 0.1µg/µl ethidium bromide inthe sample loading buffer. Promega’s system consistently yielded an average of100µg. The competitor’s system yielded an average of 70µg with wide variability.

High-quality RNA from the SV Total RNAisolation system ensures success in

subsequent experiments such asmicroarrays.

Schematic of the SV Total RNA Isolation System protocol.

(a) The PCR process is covered by patents issued and applicable in certain countries*. Promega does notencourage or support the unauthorized or unlicensed use of the PCR process. Use of this product isrecommended 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. InEurope, 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 otherpending and issued patents, which claim vectors encoding a portion of human placental ribonuclease inhibitor,are exclusively licensed to Promega Corporation.

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A.

B.

Exp. 1 Exp. 2 Exp. 30123456789

10

Inte

rslid

e CV

(%)

High degree of experimental reproducibility using RNA purified by the SV Total RNAIsolation System. Panel A. Subgrids from four representative 4K human cancerarrays (provided by Corning Incorporated) are shown. Labeled cDNA was generatedwith the ChipShot™ components of the Pronto!™ Plus System using 5ug of total RNApurified using the SV Total RNA System. Panel B. Reproducibility was assessed bydetermining 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 aresult of high quality input total RNA.

39

Tota

l RNA

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 than1 hour.

• A unique design: The novel vacuum manifold eliminates wastehandling, and the novel plate design prevents cross-contaminationduring sample processing.

• Confidence in your results: A DNase I treatment step is includedin the protocol to ensure removal of genomic DNA contamination.There is no detectable DNase carryover into the final RNApreparation.

• An applications-tested system: Tested to ensure purified RNAperforms optimally in RT-PCR.


SV 96 Total RNA Isolation System

34

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Automated isolation of total RNA using the SV 96 Total RNA Isolation System on theBeckman Coulter Biomek® FX.

Product Size Cat.#SV 96 Total RNA Isolation System 1 x 96 each Z3500

5 x 96 each Z3505For Laboratory Use.

Total RNA can be purified from 96 samplesat once in less than an hour.

The SV 96 Total RNA Isolation System provides consistent and reliable recovery ofhigh quality total RNA. Panel A: RNA yields from several cell lines using 1 x 105 cellsis consistently 0.3µg or more.

0.1

HeLa NIH3T3 CHO0

0.2

0.3

0.4

0.5

0.6

0.7

µg R

NA

SV 96SV

3467

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0.8

40

High-throughput 96-well or 384-well purification ofhighly concentrated total RNA

The MagneSil Total RNA mini-Isolation System provides:

• A simple, fast method: Elution volumes as low as 15µl giveconcentrated RNA. Only 30 minutes to process one 96-well plateand 50 minutes to process a single 384-well plate on a BeckmanCoulter Biomek® FX laboratory workstation.

• Confidence in your results: A DNase I treatment step is includedin the protocol to ensure removal of genomic DNA contamination.

• A performance-tested system: RNA rigorously tested in end-pointRT-PCR and quantitative, real-time RT-PCR.

• Efficiency: Save labor with a walk-away high-throughput 96-wellpurification automated method. 96-samples processed in only 30minutes.

• Designed for screening protocols: Total RNA isolation from smallsample sizes (up to 105 cultured cells, 20mg tissue or 20µl wholeblood/well in a 96-well format, and up to 103 cultured cells or 5µlwhole blood/well in a 384-well format).


MagneSil® Total RNA mini-Isolation System

4316

MB

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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.

Overview of the MagneSil Total RNA mini-Isolation System protocol.

Product Size Cat.#MagneSil® Total RNA mini-Isolation System 4 plates Z3351For Laboratory Use.

Tota

l RNA

Yield of total RNA from HeLa cells using the MagneSil Total RNA mini-Isolation System. TotalRNA was isolated from 1 x 105 HeLa cells/well using either the MagneSil Total RNA mini-IsolationSystem or a silica membrane based purification method. Total RNA concentration and yield werecalculated using RiboGreen® RNA Quantitation Reagent (Molecular Probes).

MagneSil Total RNA mini-Isolation System: titration of elution volume. Total RNA wasisolated from 2 x 104 HeLa cells/well using the MagneSil Total RNA mini-IsolationSystem, and eluted in various volumes from 50µl down to 10µl. Yield and concentrationobtained for each elution volume are shown. Total RNA concentration was calculatedusing RiboGreen® RNA Quantitation Reagent (Molecular Probes).

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 fromRNA purified from various titrations of HeLa cells. Twenty microliter aliquots of totalRNA isolated from a dilution series of HeLa cells in a 96-well plate were used astemplate 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 microliteraliquots (n=1) of the RT reaction were used for PCR of a c-myc target. GAPDH signaland c-myc signal were detected from as little as 10 cells.

MagneSil® RNA silica membrane

silica membrane

00.51.01.52.02.53.0

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Yield

µg

MagneSil® RNA0

10

20

30

40

50

60 Concentration

ng/µ

l

010 15 20 25 30 35 40 45 50

10

20

30

4377

MA

11_3

A

Elution Volume (µl)

Conc

entra

tion

(ng/

µl)

0.010 15 20 25 30 35 40 45 50

0.2

0.4

0.6

Elution Volume (µl)

Yiel

d (µ

g)

4378

TC

01 10 10

01,0

00

10,00

0

100,0

00

1E+06

10

20

30

40

Ct

c-myc

Cell Number

Cell Number

01 10 10

01,0

00

10,00

0

100,0

00

1E+06

10

20

30

40

Ct

GAPDHA.

B.

41

42

High-throughput, mRNA purification for sensitiveapplications

The PolyATtract Automated System provides:• Your choice of starting material: Linear recovery of poly(A)+ RNA

from 0.31–20µg of eukaryotic total RNA. Alternatively, work with0.02–5mg of eukaryotic tissue in lysate or 102–106 of culturedmammalian cells.

• Strong, selective mRNA binding: A magnetic-based purificationmethod for mRNA capture that is based on hybridization ofpoly(A)+ mRNA to a biotinylated oligo(dT) primer. The boundmRNA is then captured by streptavidn-coated paramagneticparticles. Removal of >99% of ribosomal RNA as judged byquantitative, real-time RT-PCR.

• Confidence with expression profiles: Expression profiles remainthe same as samples from total RNA and poly(A)+ RNA.

• Freedom from well-to-well cross-contamination: No detectablewell-to-well contamination as judged by RT-PCR.

• Efficiency: Save labor with walk-away automation of mRNApurification directly from cultured cells, tissue lysates or purifiedtotal RNA.

• Hassle-free automated methods: Automated methods andsupport information available at www.promega.com/automethods/

PolyATtract® Automated System

Product Size Cat.#PolyATtract® Automated System 4 x 96 Z5671For Laboratory Use.

Detection of Ten copies of GAPDH and c-myc using real-time RT-PCR analysis ofRNA purified using the MagneSil Total RNA Mini-Isolation System. To measurelinearity and sensitivity of RNA purification, GAPDH (abundant mRNA) and c-myc (raremRNA) were amplified in real-time RT-PCR from RNA purified form various titrationsof HeLa cells. Twenty microliter aliquots of total RNA isolated from a dilution series ofHeLa cells in a 96-well plate were used as template in 100_l reverse transcriptionreactions. Panel A: Five microliters aliquots (n=3) of the RT reaction were used forPCR of a GAPDH target. Panel B: Five microliter aliquots (n=1) of the RT reaction wereused for PCR of a c-myc target.

0 10 20 30 40

4073

TA

03_3

A

A.

B.

Cycle

GAPDH Target

mRNA From Cells (Avg. Ct = 23.8)

Total RNA (Avg. Ct = 23.0)

mRNA From Total RNA (Avg. Ct = 24.9)

18S rRNA Target

mRNA From CellsmRNA From Total RNA

1:10 Total RNA1:100 Total RNA

1:1,000 Total RNA1:10,000 Total RNA

10–1

100

101

0 10 20 30 40

Cycle

10–2

10–1

100

101

43

Tota

l RNA

Scalable, flexible mRNA purification for allapplications

The PolyATtract mRNA System provides:• Speed: mRNA isolation is completed in 45 minutes or less.

• Strong, selective mRNA binding: A magnetic-based purificationmethod for mRNA capture that is based on hybridization ofpoly(A)+ mRNA to a biotinylated oligo(dT) primer. The boundmRNA is then captured by streptavidin-coated paramagneticparticles. Removal of >99% of ribosomal RNA as judged byquatitative, real-time RT-PCR.

• Your choice of starting material: Configurations available for largeor small amounts of cells or tissues. mRNA isolation from totalRNA in low throughput format (PolyATtract Systems I-IV) ordirectly from crude cell or tissue lysates in scaleable format(PolyATtract System 1000).

• Convenience and ease-of-use: mRNA isolation accomplishedeasily, with a simple, magnetic-based procedure.

• Applications tested: Highly pure mRNA suitable for molecularbiology applications such as in vitro translation, cDNA synthesis,PCR, RNase protection, primer extension and Northern blots.

PolyATtract® mRNA Isolation Systems

Product Size Cat.#PolyATtract® System 1000 with Magnetic Stand Scalable Z5420PolyATtract® System 1000 without Magnetic Stand Scalable Z5400PolyATtract® 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 Z5300PolyATtract® mRNA Isolation System IV (Refill for Z5300) 15 isolations Z5310 For Laboratory Use.

Average Yields of Poly(A)+ RNA Using PolyATtract System 1000.

Size of Yield of µg mRNA/Sample Sample mRNA A260/A280 g sample

Liver 1,000mg 220µg 2.08 220Kidney 420mg 138µg 2.10 328Spleen 390mg 282µg 2.15 72Pancreas 400mg 148µg 2.11 370Lung 160mg 16µg 2.09 100NIH3T3 6 × 107 cells 20µg 2.12 200

(a)U.S. Pat. No. 5,693,784.(b)U.S. Pat. No. 6,218,531, Australian Pat. No. 745185 and other patents pending.

44

Product Size Cat.#RNAgents® Total RNA Isolation System* Scalable Z5110RNAgents® Denaturing Solution* 120ml Z5651

Product Size Cat.#SV Total RNA Isolation System(a,b)* 10 preps Z3101

50 preps Z3100250 preps Z3105

SV RNA Red Blood Cell Lysis Solution* 200ml Z3141SV RNA Lysis Buffer* 50ml Z3051

Product Size Cat.#SV 96 Total RNA Isolation System(a)* 1 x 96 each Z3500

5 x 96 each Z3505SV RNA Lysis Buffer* 50ml Z3051SV RNA Wash Solution* (concentrated) 58.8ml Z3091Nuclease-Free Water* 150ml P1195

Product Size Cat.#MagneSil® Total RNA mini-Isolation System(a)* 4 plates Z3351

Product Size Cat.#PolyATtract® Automated System* 4 x 96 Z5671Nuclease-Free Water* 150ml P1195Biotinylated Oligo(dT) Probe (50pmol/µl)* 35µl Z5261Steptavidin MagneSphere Paramagnetic Particles* 25ml Z5482PolyATtract® GTC Extraction Buffer* 120ml Z5531

Product Size Cat.#PolyATtract® System 1000 with Magnetic Stand(c)* Scalable Z5420PolyATtract® System 1000 without Magnetic Stand(c)* Scalable Z5400

Product Size Cat.#PolyATtract® mRNA Isolation System II withwith Magnetic Stand* 3 isolations Z5200PolyATtract® mRNA Isolation System I*(Refill for Z5200) 3 isolations Z5210PolyATtract® mRNA Isolation System III withwith Mangetic Stand* 15 isolations Z5300PolyATtract® mRNA Isolation System IV*(Refill for Z5300) 15 isolations Z5310


Printed in USA 09/0411268-BR-PU

Part #BR106

Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA • Telephone 608-274-4330 • Fax 608-277-2601©2004 Promega Corporation. All Rights Reserved.Prices and specifications subject to change without prior notice.


Product Size Cat.#Vac-Man® Laboratory Vacuum Manifold, 20-sample capacity 1 each A7231Vacuum Adapters 20 each A1331Vac-Man® 96 Vacuum Manifold 1 each A2291Wizard® SV 96 Binding Plates 10 pack A2271

100 each A2278MagneSil® Magnetic Separation Unit 1 each A2231MagnaBot® 96 Magnetic Separation Device 1 each V8151Heat Transfer Block 1 each Z3271MagnaBot® Spacer 1/8 inch 1 each V8581MagnaBot® Spacer 1 each V83811/4 inch Foam Spacer 1 each Z3301PolyATtract® System 1000 MagneticSeparation Stand 1 each Z5410Cat.# P1195, Z5261, Z5482, Z5531 Cat.# A2271, A2278, Z3051, Z3091, P1195. For Laboratory Use.

(a)U.S. Pat. No. 6,218,531, Australian Pat. No. 745185 and other patents pending. (b)Australian Pat. No. 730718 and other patents and patents pending.(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.

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