Genomics Workflow

RNA Purification

Plasmid DNA Purification

Genomic DNA Purification

Intergrated Extraction and Amplification

Whole Transcriptome Amplification

Whole Genome Amplification

Quantitative PCR

From DNA purification through amplification and quantification, Sigma-Aldrich offers a complete Genomics workflow solution.

FOR LIFE SCIENCE RESEARCH

Innovative Solutions for Genomics Research

Volume 3 Number 1

sigma-aldrich.com

Delivery!

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MISSION TRC shRNA provides:

n Delivery Solution – Lentiviral system for transduction of

most mammalian cells, including primary and non-dividing cells

n Comprehensive Coverage – 15,000 human and 15,000 mouse genes – 3-5 clones per gene

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Our Innovation, Your Research — Shaping the Future of Life ScienceMISSION® is a registered trademark belonging to Sigma-Aldrich Co. and its affiliate Sigma-Aldrich Biotechnology LP.

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Table of Contents

Innovative Solutions for Research

Introduction ................................................... 1

Genomics Workflow ...................................... 2

RNA Purification ............................................ 3

Plasmid DNA Purification .............................. 7

Genomic DNA Purification .......................... 13

Intergrated Extraction and Amplification ... 14

Whole Transcriptome Amplification ............ 17

Whole Genome Amplification ..................... 18

Quantitative PCR ......................................... 22

FOR LIFE SCIENCE RESEARCH

2008Volume 3Number 1

1

Sigma-Aldrich is committed to providing innovative solutions for Genomics research. Sigma has the most comprehensive offering of nucleic acid purification, amplification and detection tools. In each of these areas, Sigma provides leading products with the highest quality, performance and utility to enhance and streamline your research. This issue of BioFiles highlights a few of Sigma’s innovative products in this area including:

n GenElute™ HP Plasmid Kits, the fastest, highest yielding plasmid isolation kits available

n Extract-and Amp™ PCR kits, the world’s first integrated extraction & amplification process for rapid blood, tissue or plant assays

n GenomePlex® Whole Genome Amplification Kits for robust and representative amplification of intact or “degraded” DNA

n TransPlex® Whole Transcriptome Amplification Kit for robust and representative amplification of intact or “degraded” RNA

n JumpStart™ Taq ReadyMix qPCR reagents for highly sensitive , reproducible real-time amplification

For more information on genomics products available from Sigma, please visit sigma.com/genomics

Introduction

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Purification Amplification Quantification

Innovative Solutions Across the Genomics Workflow

Plasmid DNA Purification Pg 7

Whole Genome Amplification Pg 18

RNA Purification Pg 3

Quantitative PCR Pg 23

Whole Transcriptome Amplification Pg 17

Genomic DNA Purification Pg 12

Intergrated Extraction and Amplification p 14

Gen

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Genomics Workflow

2000+ New Products supporting Genomics, Functional Genomics, Cell Biology, Gene Expression, Proteomics, and Cell Culture

New! Life Science Web Tool Guide – learn about the optimal online tool for fi nding the products and technical information you need

Introducing the New BioUltra Proteins – risk-free, high-purity proteins, including Proteinase K, Alkaline Phosphatase and Leupeptin

To request your copy, visit sigma.com/sigmacat1

Your work is unique...innovative...groundbreakingThe New 2008-2009

Sigma® Life Science Catalog is the perfect match

sigma-aldrich.com

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GenElute™ Mammalian Total RNA Miniprep Kits

For isolation of total RNA from mammalian cells and tissues

The GenElute Mammalian Total RNA Miniprep Kit combines silica-membrane technology with a convenient spin column format for a rapid bind, wash, and elute method to prepare high quality total RNA.

Features and Benefitsn Purifies total RNA from up to 107 cells or 40 mg of tissue

per prep

n Yields up to 150 mg of pure, concentrated total RNA per prep

n Recover RNA from as few as 100 cells

n Simple and efficient – 12 to 18 preps in 30 minutes

n Faster than gravity flow anion exchange methods

n No cumbersome steps associated with resins and magnetic slurries

n 40% more purifications than the leading supplier

Storage: Room Temperature R: 24-20/22-41-37/38 S: 53-45-26-36/37/39

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High Quality RNA from various tissues and cellsFigure 1A. Formaldehyde-agarose gel and Northern blot of total RNA purified with GenElute™ Mammalian Total RNA Miniprep Kit.Upper panel: 2 mg of each RNA analyzed on a 1.2% agarose gel containing 0.6 M formaldehyde.Lower panel: Corresponding Northern blot hybridized with a 32P-labeled RNA probe for GAPDH in PerfectHyb™ Plus hybridization buffer (Cat. No. H7033).Note: The GAPDH probe detected a single mRNA band in every lane with little or no smearing. Even RNA from pancreas, which is known to have high RNase levels, is not degraded.

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Ordering Information

Cat. No. Product Description Preps Quantity

RTN10 GenElute™ Mammalian Total RNA Miniprep Kit 10 1 kit

RTN70 GenElute™ Mammalian Total RNA Miniprep Kit 70 1 kit

RTN350 GenElute™ Mammalian Total RNA Miniprep Kit 350 1 kit

RNA Purification

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GenElute™ mRNA Miniprep Kits

High yield isolation of mRNA from mammalian cells, tissues, or total RNA

Procedures such as cDNA synthesis, expression profiling and others require separation of mRNA from the vastly more abundant rRNA and tRNA. The GenElute mRNA Kits provide convenient procedures for isolating polyadenylated mRNA from previously prepared total RNA or directly from mammalian cells and tissues. For direct mRNA preparation, cells or tissues are disrupted with SDS/proteinase K digestion to release RNA and eliminate RNases. Both kit types use oligo dT30 covalently linked to 1 mm polystyrene beads to capture polyadenylated mRNA by hybridization. The polystyrene beads

remain suspended during hybridization, eliminating the need for mixing or rocking, as is common for cellulose or magnetic particles. Polystyrene was also chosen because oligo (dT) polystyrene beads yield cleaner mRNA with fewer stringent washing steps than does the more commonly used oligo (dT) cellulose (2 or 3 wash steps versus 10 or more). With the GenElute Kits, mRNA-bead complexes are washed on a microcentrifuge spin filter, and eluted into 10 mM Tris-HCL, pH 7.5. mRNA prepared with either kit is suitable for a variety of downstream applications such as Northern blotting, Expression Array or Chip Hybridizations, and cDNA Synthesis and Library Construction.

Features and Benefitsn mRNA captured on oligo (dT) polystyrene beads in 10 minutes,

with no mixing or rocking (Figure 1)

n Poly (A)+ mRNA isolated from total RNA in 40 minutes or 80 minutes directly from cells and tissues (Figure 1)

n Oligo (dT) polystyrene beads require fewer wash steps

Storage: Room Temperature MRN10, MRN70; R: 61-64-62-22-36/37 S: 53-45-36/37/39-23 DMN10, DMN70; R: 22-24-26-36/37 S: 22-24-26-36/37

Preparation Time

from total RNA direct from cells or tissues

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Figure 1. Comparison of the preparation time, to isolate mRNA from total RNA or direct from cells or tissues, using GenElute™ mRNA Miniprep Kit and other commercially available kits. Each kit was prepared according to the procedure supplied by the vendor.

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Figure 2. Northern blot comparison of mRNA prepared directly from cells and tissues with GenElute Direct mRNA Miniprep Kit and competitor kits. mRNA was prepared from 25-35 mg of mouse liver with Sigma’s GenElute Direct mRNA Miniprep Kit or several commercially available direct mRNA miniprep kits. A portion of each mRNA preparation equal to the amount of 10 mg of liver was evaluated by Northern blot hybridization with 32P-labeled RNA probes. Hybridization was detected and quantitated by scanning the blots with a Perkin Elmer Instant Imager. Hybridization signals from each lane on the northern blot, expressed as a percent of the maximum signal for that probe, are plotted in the accompanying graphs.

Ordering Information

Cat. No.Product Description

Preps/ Kit Starting Material

MRN10 GenElute™ mRNA Miniprep Kit

10 5-500 mg total RNA

MRN70 GenElute™ mRNA Miniprep Kit

70 5-500 mg total RNA

DMN10 GenElute™ Direct mRNA Miniprep Kit

10 Up to 10 mammalian cells or 50 mg tissue

DMN70 GenElute™ Direct mRNA Miniprep Kit

70 Up to 107 mammalian cells or 50 mg tissue

Ordering Information

Cat. No.Product Description

Preps/ Kit Starting Material

MRN10 GenElute™ mRNA Miniprep Kit

10 5-500 mg total RNA

MRN70 GenElute™ mRNA Miniprep Kit

70 5-500 mg total RNA

DMN10 GenElute™ Direct mRNA Miniprep Kit

10 Up to 107 mammalian cells or 50 mg tissue

DMN70 GenElute™ Direct mRNA Miniprep Kit

70 Up to 107 mammalian cells or 50 mg tissue

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TRI Reagent® RNA Isolation ReagentFor isolation of total RNA from a variety of starting materials TRI Reagent® is an improved version of the single-step total RNA isolation reagent developed by Chomczynski.1 The RNA isolation method based on this reagent is widely recognized and proven for RNA applications and is supported by a substantial publication list.2

It is ideal for quick, economical, and efficient isolation of total RNA or the simultaneous isolation of RNA, DNA, and proteins from samples of human, animal, plant, yeast, bacterial and viral origin.

Features and Benefits

n Easily scalable RNA isolation

n Works with many sources: human, plant, yeast, bacterial, or viral

n Better yields than traditional guanidine thiocyanate/cesium chloride methods

n Three convenient formulations of TRI Reagent®

Storage: 2-8 °C R: 23/24/25-32-34-48/20/21/22-54-53-68 S: 26-36/37/39-45-61

TRIPu

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Figure 1. Total RNA was prepared from HeLa cells using TRI Reagent® from Sigma and equivalent reagents from other various suppliers Figure 1. Total RNA from HeLa cells was prepared using TRIPure®, Sigma TRI Reagent®, and TRIzol®. An aliquot of total RNA was analyzed on a 1% agarose gel. RNA Marker (M) ranged from 0.2 bp-10 kb (Cat. No. R7020).

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Table 1. TRI Reagent® FormulationsProduct Name Sample Type Sample Volume TRI Reagent® Volume TRI Reagent® Tissues, cultured adherent cells,

cell pelletsup to 100 mg tissue, 107 cells, or 102 cm plate area

1 mL

TRI Reagent® BD Whole blood, plasma, serum 0.25 mL blood derivatives 0.75 mL

TRI Reagent® LS Cell suspension, CSF, amniotic fluid 0.25 mL fluid samples 0.75 mL

Table 2. Typical RNA Yield

Tissue Yield (mg RNA/mg tissue)

Liver 6-10

Spleen 6-10

Kidney 3-4

Skeletal Muscle

1-1.5

Brain 1-1.5

Placenta 1-4

Cell Yield (mg RNA/106 cells)

Epithelial 8-15

Fibroblast 5-7

Ordering Information

Cat. No. Product Description Quantity

T9424 TRI Reagent® RNA, DNA and Protein Isolation Reagent

25 mL

100 mL

200 mL

6 x 100 mL

T3809 TRI Reagent® BD 25 mL

100 mL

200 mL

T3934 TRI Reagent® LS 25 mL

100 mL

200 mL

References1. Chomczynski, P. and Sacchi, N., Single-step method of RNA

isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156 (1987).

2. Chomczynski, P. and Mackey, K., Short Technical Reports. Modification of the TRI Reagent® procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. BioTechniques 19: 924-945 (1995).

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GenElute™ HP Mini, Midi, and Maxiprep Plasmid KitsPlasmid DNA from a maxiprep in less than 30 minutes

The GenElute HP Plasmid Purification kits yield high quality plasmid DNA in less than 30 minutes (Figure 1). GenElute HP Plasmid kits feature a filter syringe for lysate clearing, and binding columns that can be used with a vacuum or spin purification format.

An overnight recombinant E. coli culture is harvested by centrifugation and subjected to a modified alkaline-SDS lysis procedure. The lysate is cleared with a filter syringe and DNA is bound to the silica-based membrane. The remaining contaminants are removed by a wash step. Finally, the bound DNA is eluted in buffer or water (Figure 2). High quality DNA is suitable for the most demanding applications including transfection (Figure 3), restriction digestion (Figure 4), ligation, automated sequencing (Figure 5).

Features and Benefits

n From harvested bacterial culture to pure plasmid DNA in 30 minutes or less

n Up to 25 mg (Mini), 350 mg (Midi), and 1.2 mg (Maxi) yield of high-copy plasmid DNA

n Offers the flexibility of a vacuum or spin format

n Contain fewer plastic components than other high speed kits, reducing the amount of waste

n No phenol/chloroform extraction or alcohol precipitation required

n Kits are stable at room temperature for convenient storage

n Cost effective

Storage: Room Temperature R: 22-36/37/38 S: 26-36

GenElute HP Plasmid Kits

Preparation time for different maxiprep plasmid purification kits

Figure 1. Comparison of time required per isolation using different Maxiprep plasmid isolation systems. Plasmid DNA was isolated following each manufacturers recommended protocol. Each system was tested multiple times and the average preparation time is shown here.

Plasmid DNA yield for different maxiprep kits

Figure 2. Comparison of plasmid DNA yield using different Maxiprep plasmid isolation systems. Plasmid DNA was isolated following each manufacturers recommended protocol. All samples were done in duplicate.

“I spend less time purifying DNA and I get higher yields with the Sigma GenElute™ HP Plasmid Purification Kits. The kits give me time back that I can spend on other experiments.”

— Rene Rijnbrank, The University of Texas Medical Branch, Galveston

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Comparison of transfection efficiencies of plasmids prepared with different plasmid isolation methods

Figure 3. Transfection of cells with plasmid isolated using different plasmid purification kits. Data shows an average of 3 replicates for each named isolation method. All transfections were in CHO-K1 cells. The OD measurements were taken at 420 nm recording the units of b-gal/plate. The cells were grown to 60-70% confluency and transfected using 3 mg of plasmid pSPORT-CMV/15 mg of ESCORT™ IV. b-Gal activity was determined 54 hours post-transfection.

The elution step can be modified for higher DNA concentration. After the addition of elution buffer, spin the column at 1000 x g for 5 minutes rather than the 3000 x g stated in the standard protocol. This will result in a reduced volume of eluate coming off the column which is approximately 3x more concentrated. This method delivers approximately 90% of the yield of the standard protocol.

Purified plasmid DNA is suitable for restriction enzyme digestions

pUC18/JM109 pBR322/HB101

S Q S Q M

Figure 4. Restriction digestion of plasmid DNA isolated using the GenElute™ HP Plasmid Maxiprep Kit and a comparable kit from Supplier Q. The plasmid DNA (800 ng) was digested with 10 units of Hind III at 37 °C for 4 hours. Each plasmid sample represents 100 ng of undigested DNA (left) and digested DNA (right) loaded in each pair of lanes. Samples were loaded on a 1% TBE agarose gel. The marker (M) used was a 1 kb DNA Ladder (Cat. No. D0428).

High Quality DNA for Automated Sequencing

Figure 5. Electropherogram revealing >700 bases of sequence from pCMV-SPORT-bgal purified from GenElute™ HP Plasmid Midiprep Kit. Cycle Sequencing was performed using 500 ng template, a T7 sequencing primer and ABI BigDye® terminator chemistry. Sequencing reactions were resolved on an ABI Prism® 377 XL instrument with a 48 cm gel cassette containing AutoPAGE™ Plus 4.5% acrylamide at 2.4 kV, 48 °C for 7 hours. (Cat. No.. P8977)

Ordering Information

Cat. No. Product Description Preps Quantity

NA0150S GenElute™ HP Plasmid Miniprep Kit 10 1 Kit

NA0150 GenElute™ HP Plasmid Miniprep Kit 70 1 Kit

NA0160 GenElute™ HP Plasmid Miniprep Kit 350 1 Kit

NA0200S GenElute™ HP Plasmid Midiprep Kit 4 1 Kit

NA0200 GenElute™ HP Plasmid Midiprep Kit 25 1 Kit

NA0300S GenElute™ HP Plasmid Maxiprep Kit 4 1 Kit

NA0300 GenElute™ HP Plasmid Maxiprep Kit 10 1 Kit

NA0310 GenElute™ HP Plasmid Maxiprep Kit 25 1 Kit

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High Yields and Low Endotoxin Levels in Less TimePurification Kit Plasmid Yield (mg) Endotoxin Levels (EU/mg) Time/Prep (minutes)

GenElute HP Endotoxin-free Megaprep Kit 6.0 1-10 75

Supplier Q Endotoxin-Free Plasmid Mega Kit 2.5 1-45 210

Supplier B Plasmid Mega EF Kit 2.0 39-46 210

Supplier M High Purity Plasmid Megaprep System 1.7 13-38 90Comparison of plasmid yield, endotoxin levels and time/prep for the different purification systems used for isolating high copy number plasmid pCMV-SPORT-s-gal/DH5a™ in LB.

GenElute™ HP Endotoxin-free Plasmid Megaprep KitIsolate endotoxin-free plasmid DNA in 90 minutes or less with the GenElute HP Endotoxin-free Plasmid Megaprep Kit. The kit features an endotoxin removal system for isolation of DNA with £0.1EU/µg. With a convenient vacuum format and no need for alcohol precipitation, this kit provides the fastest method for isolation of endotoxin-free plasmid DNA.

Features and Benefits

n Fast – from harvested bacterial culture to pure plasmid DNA in 90 minutes

n Convenient – vacuum format with no ethanol precipitation required

n High Yields – 5 mg of high-copy plasmid DNA (£0.1EU/µg)

n Economic – lower cost, higher yields and significantly reduced labor equals substantial savings — in both dollars and time

Storage: Room Temperature R: 10-22-36/37/38-48/20-67 S: 26-36/37/39-45

“I switched to Sigma’s GenElute™ HP Plasmid Purification Kits because the yield is much higher, the purity is better or equal to my previous method and, importantly, it takes me only a quarter of the time.”

— Waldemar Popik, Johns Hopkins University

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Transfect Endotoxin-Sensitive Cell Lines

Figure 2. Comparison of transfection efficiencies of high-copy plasmid DNA pCMV-SPORT-b-gal/DH5a™ into endotoxin sensitive cell line HuH-7 cells and non-endotoxin sensitive cell line CHO-K1 cells. The data shows the average and standard deviations of three replicates for both cell lines evaluated. Untransfected HuH-7 and CHO-K1 cells represent cells treated with transfection reagents without any plasmid DNA.

Ordering Information

Cat. No. Product Description Preps Quantity

NA0600 GenElute™ HP Endotoxin-free Plasmid Megaprep Kit

5 1 kit

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GenElute™ HP Select Plasmid Gigaprep KitThe GenElute HP Select Plasmid Gigaprep Kit offers a simple, rapid, and cost-effective method for isolating endotoxin-free plasmid DNA from recombinant E. coli cultures. Yields of 15 mg of high copy plasmid DNA with £0.1 EU/mg can be recovered from E. coli cultures. High-quality, endotoxin-free DNA can be used for the most demanding applications including transfection with endotoxin-sensitive cells and gene therapy research.

Procedure: An overnight recombinant E. coli culture is harvested by centrifugation and subjected to a modified alkaline-SDS lysis. The lysate is cleared by filtration followed by the addition of a binding solution optimized for endotoxin-free plasmid preparations. The plasmid DNA is then captured onto a silica membrane in the presence of high salts, while endotoxins are prevented from adsorbing to the membrane. Contaminants are removed with three wash steps. Finally, the bound DNA is eluted in endotoxin-free water.

Application: High-quality, endotoxin-free DNA is ready for immediate use for the most demanding applications includ-ing transfection with endotoxin-sensitive cells and gene therapy research.

Features and Benefits

n Sophisticated — utilizes HP Select technology, the premier plas-mid purification method

n Novel — includes endotoxin removal system

n High Yields — 15 mg of high-quality, endotoxin-free (£0.1EU/mg) plasmid DNA in 2 hours or less

n Convenient — vacuum format with no ethanol precipitation required

n Versatile — can be used to purify low-, medium-, and high-copy plasmid DNA

Storage: Room Temperature R: 10-22-36/37/38-48/20-67 S: 53-26-36/37/39

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More Plasmid DNA in Significantly Less Time

Purification System Plasmid Yield (mg) Time/Prep (hours) Alcohol Precipitation Required?

GenElute HP Select Plasmid Gigaprep Kit 12.2 1.5 NO

Supplier Q Endo-Free Plasmid Giga Kit 10.9 4.8 YES

Supplier B Plasmid Giga EF Kit 6.3 3.3 YES

Comparison of plasmid yield and time/prep for the different purification systems used for isolating medium-copy number plasmid pFLAG-CMV1/DH5a in LB medium.

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Isolate DNA from Low-, Medium-, and High-copy Plasmids

Figure 1. Maximum plasmid DNA yield obtained with different constructs using GenElute HP Select Plasmid Gigaprep Kit.

The lysate clearing agent functions as a prefilter, eliminating the need for an extra step when preparing cleared lysates. Just add the clearing agent to the lysate, mix thoroughly, wait five minutes, and filter.

Transfect Endotoxin-Sensitive Cell Lines

Figure 2. Comparison of transfection efficiencies of high copy plasmid DNA pCMV-SPORT-b-gal/DH5a™ into endotoxin sensitive cell line HuH-7 cells. The data shows the average and standard deviations of three replicates for the cell line evaluated. Untransfected HuH-7 represent cells treated with transfection reagents without any plasmid DNA.

Ordering Information

Cat. No. Product Description Preps Quantity

NA0800 GenElute™ HP Select Plasmid Gigaprep Kit

5 1 kit

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Are you starting a new lab?

Are you moving to a new location?

Have you received your first research grant?

If you have answered “yes” to any of the above questions, let us help you get your new lab set up in an easy, effective and economical way with the Sigma-Aldrich New Lab Start-Up Program.

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Sigma-Aldrich New Lab Start-Up Program

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GenElute™ Mammalian Genomic DNA Miniprep KitFor purification of genomic DNA from a variety of mammalian sources

The GenElute Mammalian Genomic DNA Purification Kit provides a simple and convenient way to isolate pure, high molecular weight DNA from a variety of mammalian sources (Figure 1 and 2). These kits use a silica-based membrane, specially selected for genomic DNA purification, in a convenient spin column format. Mammalian cells and tissues are lysed with a chaotropic salt-containing buffer to ensure denaturation of macromolecules. DNA is bound to the spin column membrane and the remaining lysate is removed by centrifugation. A filtration column is used to remove cell debris. After washing to remove contaminants, the DNA is eluted with buffer into a collection tube. The purified DNA may be used in many applications such as sequencing, cloning, blotting, restriction digestion, ligation, and PCR.

Features and Benefits

n Typical DNA yields of 25 mg from 2 x 106 cultured cells or 30 mg from 25 mg of tissue (Table 1)

n Preparation time is only 20 minutes after lysis

n Purified genomic DNA has A260/A280 ratios between 1.6 and 1.9

n No need for mechanical homogenization

n 40% more purification preps offered than other leading supplier

Storage: Room Temperature R: 20/21/22 S: 26-36

Table 1. Yields Produced Using GenElute™ Mammalian Genomic DNA Purification Kits*

Material Amount Typical Yield

Jurkat Cells (human) 2 x 106 cells 5-10 mg

HEK293 Cells (human) 2 x 106 cells 10-20 mg

HeLa Cells (human) 2 x 106 cells 15-25 mg

Mouse Pancreas Tissue 20 mg 10-25 mg

Mouse Spleen Tissue 10 mg 10-25 mg

Mouse Thymus Tissue 16 mg 10-25 mg

Mouse Lung Tissue 20 mg 5-15 mg

Mouse Brain Tissue 16 mg 5-15 mg

Mouse Kidney Tissue 20 mg 10-25 mg

Mouse Liver Tissue 25 mg 10-30 mg

*With RNase and Proteinase K treatment

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Genomic DNA purified from tissues using GenElute™ Mammalian Genomic DNA Miniprep Kit

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Figure 1. Genomic DNA purified from mammalian tissues. Purified genomic DNA, from the indicated mouse tissues, was isolated with the GenElute™ Mammalian Genomic DNA Miniprep Kit. Genomic DNA (200 ng/lane) was analyzed on a 0.8% agarose gel to illustrate yield and integrity. Markers are lambda DNA digested with Hind III. (Cat. No. D9780)

Yield Comparison

Sigma Supplier Q30

25

20

15

10

5

0Liver

25 mgKidney20 mg

Lung20 mg

Pancreas20 mg

Spleen16 mg

Thymus16 mg

Mouse Tissue (mg)

Figure 2. Yields of genomic DNA compared with Supplier Q using 2 x 200 µL elutions.

Save time with the Genomic DNA Miniprep Kit! Unlike most kits on the market, the elution buffer in our Sigma kit does not have to be heated.

Ordering Information

Cat. No. Product Description Preps Quantity

G1N10 GenElute™ Mammalian Genomic DNA Miniprep Kit

10 1 kit

G1N70 GenElute™ Mammalian Genomic DNA Miniprep Kit

70 1 kit

G1N350 GenElute™ Mammalian Genomic DNA Miniprep Kit

350 1 kit

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Extract-N-Amp™ Tissue PCR KitsFrom tissue or cells to PCR in 15 minutesThe Extract-N-Amp Tissue PCR Kits provide all the reagents necessary to rapidly extract DNA from a wide variety of cells and tissues and amplify targets of interest by PCR (Fig. 1). A novel extraction method eliminates the need for long enzymatic digestions or homogenization. The kit also includes a specially formulated hot start

PCR ReadyMix™ for amplification directly from the extract. The PCR ReadyMix comes in two formulations: Extract-N-Amp PCR ReadyMix and REDExtract-N-Amp™ PCR ReadyMix. The REDExtract-N-Amp PCR ReadyMix contains an inert dye that acts as a tracking dye and allows for convenient loading of PCR reactions onto agarose gels for analysis.

The kit comes with validated protocols to extract and amplify genomic DNA from mouse-tails, hair, animal tissue, saliva, and buccal swabs. In a typical procedure, genomic DNA is extracted from a tissue sample that has been incubated in the tissue preparation solution and extraction solution for 10 minutes at room temperature. The sample is heated to 95 °C for 3 minutes and then mixed with a third solution to neutralize inhibitory substances prior to PCR. A portion of the DNA extract is then added to a PCR reaction containing primers and either the REDExtract-N-Amp or Extract-N-Amp PCR ReadyMix included in the kit.

Feature and Benefits

n Fast — rapid extraction of genomic DNA for PCR in 15 minutes

n Convenient — no long enzymatic digestions

n Practical — perfect for quick genomic DNA isolation for genotyping

n Flexible — protocols available for mouse-tails, hair, animal tissue, saliva, and buccal swabs

n Specific — hot start antibody for highly specific PCR amplification of genomic DNA Storage: –20 °C Shipped in wet ice R: 36/37/38-42/43 S: 26-36

M M

2 kb —

1.5 kb —

1 kb —

750 kb —

500 kb —

300 kb —

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Figure 1. PCR analysis of genomic DNA extracted from various samples using Sigma’s Extract-N-Amp™ Tissue PCR Kit. Genomic DNA was extracted from the samples using the appropriate protocol as described in the Extract-N-Amp Tissue Technical Bulletin. In all cases, the extraction procedure was com-pleted in less then 15 minutes, then DNA was amplified using hot start PCR ReadyMix™. The products generated are 1181 bp for the Interleukin 1 Beta gene in mouse and 1820 bp for the Carnitine palmitoyltransferase II in human. Markers are PCR Marker (Cat. No. P9577).

Tissue Sample +Tissue Preparation Solution

Incubate with Extraction Solutionfor 10 minutes at room temperature.

Heat at 95 °C for 3 min.

Add Neutralization Solution.

Mix aliquot withREDExtract-N-Amp™or Extract-N-Amp™

PCR ReadyMix™ and primers.

Transfer to thermal cycler.

PCR

Directly load amplified PCRproduct on gel.

Overview of Extract-N-Amp™Tissue PCR Kit Procedure

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Integrated Extraction and Amplification

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Stability of Mouse-Tail Extracts at 37 °C

t=0 6 wk, 37 °C

3 wk, 37 °C 2 mo, 37 °C

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Figure 3. Mouse-tail samples were extracted according to the procedure in the Technical Bulletin for the Extract-N-Amp™ Tissue PCR Kit. 4 mL aliquots were analyzed immediately by quantitative PCR with SYBR® Green detection on an ABI Prism® 7700. DNA standards for quantitative PCR were purified DNA prepared from mouse tails using the GenElute Mammalian Genomic DNA kit (Cat. No. G1N70) and stored as single use aliquots at –20 °C. The mouse-tail extracts were stored at 37 °C (accelerated storage). Quantitative PCR was repeated after 3 weeks, 5 weeks and 2 months from extracts at 37 °C. Results for storage at 37 °C are shown. These results sug-gest that extracts will be stable for at least 6 months at the recommended storage temperature of 4 °C.

Ordering InformationCat. No. Product Description Extractions AmplificationsXNATS RedExtract-N-Amp™

Tissue PCR Kit10 10

XNAT RedExtract-N-Amp™ Tissue PCR Kit

100 100

XNATR RedExtract-N-Amp™ Tissue PCR Kit

1000 1000

XNAT2 Extract-N-Amp™ Tissue PCR Kit

100 100

XNAT2R Extract-N-Amp™ Tissue PCR Kit

1000 1000

Inquire for bulk and high-throughput needs.

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Figure 2. Sequence determination for 1181 bp Interleukin 1 Beta mouse-tail PCR product. The DNA extraction and PCR were performed using Sigma’s Extract-N-Amp™ Tissue PCR Kit. The PCR product was purified with the GenElute™ PCR Clean-up Kit (Cat. No. NA1020). The product was sequenced directly using BigDye® terminator v 3.1 chemistry. Sequencing reactions were analyzed on an ABI 3730xl.

Visit our web site sigma.com/xnaffpe for protocol modifications, including FFFPE tissue.

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Transfer to thermal cycler.

Add aliquot of extract &Mix aliquot with

REDExtract-N-Amp™PCR Reaction Mix and primers.

For fresh or frozen blood samples, monolayers

Extract-N-Amp Blood PCR Kit

Remove aliquot of blood

Mix blood with Lysis Solution

Add Neutralization Solution

Incubate room temp. 5 min.

Directly load amplified PCR product on gel.

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Extract-N-Amp Blood PCR KitsFrom whole blood to PCR in 8 minutesExtract-N-Amp Blood PCR Kits contain all of the reagents necessary to rapidly extract genomic DNA from whole blood and amplify targets of interest by PCR. This novel extraction system eliminates the need for any type of purification, organic extraction, centrifugation, heating, filtration or alcohol precipitation. The kit also includes a PCR ReadyMix™, formulated for amplification directly from the extract. This formulation uses an antibody based hot-start for specific amplification.

Features & Benefits

n Efficient 8 minute prep allows greater speed and higher throughput

n No need for purification, organic extraction, centrifugation or alcohol precipitation

n Simple procedure makes it perfect for single tube or high throughput applications

n Easily automatable procedure

n Stable extract can be aliquotted and used for up to 50 amplifi-cations.

n Can be used with whole blood or blood cards

Ordering InformationCat. No. Product Description Extractions AmplificationsXNAB2 Extract-N-Amp™ Blood

PCR Kit 100 100XNAB2R Extract-N-Amp Blood

PCR Kit 1000 1000XNAB2RE Extract-N-Amp Blood

PCR Kit 1000 5000XNABS REDExtract-N-Amp™

Blood PCR Kit 10 10XNAB REDExtract-N-Amp

Blood PCR Kit 100 100XNABE REDExtract-N-Amp

Blood PCR Kit 100 500XNABR REDExtract-N-Amp

Blood PCR Kit 1000 1000XNABRE REDExtract-N-Amp

Blood PCR Kit 1000 5000

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TransPlex® Whole Transcriptome Amplification KitThe TransPlex WTA Kit enables representative amplification of total RNA in less than 4 hours without 3’-bias. Micrograms of amplified DNA products suitable for both qPCR and microarray analyses can be generated from nanograms of starting RNA isolated from various sources such as blood, tissue biopsy, cultured cells, fixed and frozen tissues, or from non-human samples such as bacteria, plants and animals.

The TransPlex WTA process has two steps; library synthesis and library amplification. To synthesize the library sample RNA is incubated with a reverse transcriptase and non-self-complementary primers comprised of a quasi-random 3’ end and a universal 5’ end. When annealed primers are extended by polymerase, displaced single strands are generated which become new templates for primer annealing and extension. This process creates an OmniPlex® library comprised of random, overlapping 100-1000 base fragments flanked by a universal end sequence. Universal-primer PCR is then used to amplify the OmniPlex® library and produce WTA products.

Features and Benefits

n Achieve 1000–10,0003 amplification in less than 4 hours with no more than 30 minutes of “hands on” time required

n Only 5 ng of total RNA is required for a sufficient template to perform microarray profiling

n TransPlex reagents and protocols are optimized to give linear representation of all expressed genes and exons

n Effectively amplifies degraded RNA, including formalin-fixed, paraffin-embedded samples for profiling

Components: WTA Library Synthesis Buffer WTA Library Stabilization Solution WTA Library Synthesis Enzyme WTA Amplification Master Mix WTA dNTP Mix Nuclease-Free Water

Storage: –20 °C Shipped in wet ice

Ordering InformationCat. No. Product Description Quantity

WTA1 TransPlex Whole Transcriptome 10 reactions Amplification (WTA) Kit 50 reactions

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WTA Procedure

WTA Procedure

Add primers

Input RNA

First Strand cDNA

Second Strand cDNA

OmniPlex Library

Amplified cDNA

Anneal and extend

Denature

PCR

QPCR or microarray analysis

qPCR Primer Set

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-4

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Unamplified Total RNA Amplified Total RNA

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qPCR Primer Set. Differential expression of seven mRNAs (cDNAs) in human liver and UHR (Universal Human Reference®) was measured by quantitative PCR. DC(t) values (UHR - Liver) were calculated for unamplified cDNA, prepared directly from RNA by reverse transcription, and amplification product prepared from 25 nanograms of total RNA with the Transplex WTA Kit. (Normalization of C(t)s for liver or brain was accomplished by subtracting the average DC(t) for all primer sets for a given tissue from each tissue-specific DC(t).) Uniformity between unamplified and amplified DC(t)s for individual mRNAs demonstrates maintenance of differential patterns of RNA expression in liver and UHR when using the Transplex WTA Kit.

Universal Human Reference Total RNA is a product of Stratagene.

Whole Transcriptome Amplification

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Move Beyond the Limitations of Traditional PCR…Unsurpassed Yield, Unlimited Potential GenomePlex® Whole Genome Amplification (WGA) products provide a robust and accurate method of amplifying nano-gram quantities of starting material into microgram yields with minimal allele drop out. GenomePlex amplified DNA is suitable for multiple downstream applications including gel electrophoresis, QPCR, CGH microarray, STR analysis, SNP analysis and sequencing.

Features and Benefits

n Choose from a variety of DNA sources: whole blood, blood card, plasma, serum, buccal swab, plant, soil, FFPE tissue, single cells.

n Complete representation of the entire genome with no detect-able allele bias

n Increase amplification accuracy with WGA DNA Polymerase

n Preserve precious source material by amplifying nanogram amounts of starting genomic DNA into microgram yields in a few hours

n Enjoy the compatibility GenomePlex provides with many down-stream applications: TaqMan® Assays, SNP analysis, microarray analysis and sequencing.

For the latest on the entire GenomePlex product line, visit sigma.com/wga

The GenomePlex TechnologyGenomePlex WGA technology is derived from a proprietary amplification method that is based upon random fragmen-tation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands generate a library of DNA fragments with defined 3’ and 5’ termini, the OmniPlex® library. This library is replicated using a lin-ear, isothermal amplification in the initial stages, followed by a limited round of geometric amplification (PCR). The standard GenomePlex reaction can be performed with minimal hands-on time and can produce amplified DNA in as little as three hours.

Fragmentation

Anneal primers

Extend

PCR amplify

2nd priming event

OmniPlex library formation

Amplified Genomic DNA

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nWhole Genome Amplification

A technical animation of the GenomePlex technology is available at biocompare.com/console/sigma/genomeplex_video.asp

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Innovative Solutions for Whole Genome AmplificationSigma-Aldrich has a dedicated team of R&D scientists committed to providing best-in-class products for whole genome amplification. We continue to expand and enhance our WGA product line to offer you a range of solutions for whole genome amplification from a variety of starting material. The WGA family of products includes:

(WGA1) GenomePlex Whole Genome Amplification Kit

(WGA2) GenomePlex Complete Whole Genome Amplification Kit

(WGA4) GenomePlex Single Cell Whole Genome Amplification Kit

(WGA5) GenomePlex Tissue Whole Genome Amplification Kit

(WGA3) GenomePlex WGA Reamplification Kit

Feature and Benefits

n The GenomePlex WGA Kit (WGA1), contains all the necessary reagents to perform fragmentation and library preparation and allows the researcher the flexibility of using their amplification enzyme of choice.

n The GenomePlex Complete WGA Kit (WGA2) includes all the reagents necessary for WGA as well as an optimized enzyme, WGA DNA Polymerase. This enzyme provides for increased accuracy in amplification, as evidenced by producing no ampli-con in the negative control reactions.

n The GenomePlex Single Cell WGA Kit (WGA4) includes all of the reagents necessary for cell lysis and whole genome amplification of genomic DNA from a single cell.

n The GenomePlex Tissue WGA Kit (WGA5) includes all of the reagents necessary for extraction of genomic DNA from FFPE, fresh or frozen tissue and whole genome amplification.

n The GenomePlex WGA Reamplification Kit (WGA3) allows multiple rounds of reamplification while maintaining genetic representation.

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Product Selection

What is your starting material?

Genomic DNA fromany source

Genomic DNA fromany source

Single cell

FFPE, frozen orRNAlater® preserved

tissue

Do you want the increased accuracy ofthe WGA Polymerase?

Do you want the flexibility of choosing your own enzyme for amplification?

Do you want to reamplify your WGA product?

WGA2

WGA4

WGA5

WGA1

WGA3

WGA1 — Whole Genome Amplification KitWGA2 — Complete Whole Genome Amplification KitWGA3 — WGA Reamplification KitWGA4 — Single Cell WGA KitWGA5 — Tissue WGA Kit

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Amplification of Genome-Representative DNA from Limited Starting Material The GenomePlex Complete Whole Genome Amplification Kit (Cat. No. WGA2) contains everything required for whole genome amplification including an optimized enzyme, WGA DNA Polymerase. The WGA DNA Polymerase provides increased accuracy in amplification, as evidenced by producing no amplicon in the negative control reactions. WGA has been used in a variety of applications, and is suitable for use with purified genomic DNA from a variety of sources including blood cards, whole blood, buccal swabs, tissue, soil, plant, and serum. GenomePlex WGA uses nanogram quantities of starting genomic DNA, which after PCR yields on average 10 µg of amplified DNA. After purification, the WGA product can be analyzed in a manner similar to any genomic or chromosomal DNA sample. A number of downstream applications may be performed including TaqMan® assays, CGH analysis, SNP analysis, and sequencing.

Achieve Robust Amplification Representative of the Original Input Genome

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

WGA was performed on increasing concentrations of human genomic DNA. Amplification product can be detected on an agarose gel with as little as 10 pg of input DNA. Optimal performance is found with 1 to 10 ng of start-ing material. Increasing the amount of input DNA to 100 ng is not recom-mended.

Lane M: DNA Marker Lanes 7,8: 100 pg DNA Lanes 1,2: no template Lanes Lanes 9,10: 1 ng DNA Lanes 3,4: 1 pg DNA Lane Lanes 11,12: 10 ng DNA Lanes 5,6: 10 pg DNA Lanes Lanes 13,14: 100 ng DNA

GenomePlex WGA was performed on genomic DNA isolated from HT29 colon carcinoma cells and from a healthy human male. 2.5 µg of WGA product was labeled with Cy™3 or Cy5 dye using the Genomic DNA Labeling Kit PLUS (Agilent). The entire labeled sample was loaded onto an Agilent Human Genome CGH Microarray 105A. Specific activities were between 28 and 43 for all samples, and always within 50% of samples being compared. The dye swaps (A & B) demonstrate that there was no bias in the DNA labeling and the aberrations detected are consistent with the HT-29 karyotype.

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Looking for a high-throughput system for the rapid and highly representative amplification of genomic DNA from trace amounts of starting material? Visit sigma.com/wgaautomation for automated WGA protocols and methods.

NEW! Technical note on Agilent Array CGH with WGA, visit sigma.com/wgacgh.

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Comparison of WGA5 Amplified & Unamplified GenElute Purified FFPE Gallbladder DNA

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Chromosome Location (Each color = different chromosome)

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eComparison of WGA5 Amplified & Unamplified GenElute Purified FFPE Gallbladder DNA. Real-time qPCR was performed targeting 96 loci on unamplified human genomic DNA isolated directly from formalin-fixed paraffin embedded (FFPE) gallbladder tissue or DNA products generated with the GenomePlex® Tissue Whole Genome Amplification Kit starting with 1 mg of the same FFPE gallbladder tissue. Fold change was calculated as 2CT, where CT is the average CT value generated from GenomePlex amplified DNA minus the average CT value generated from unamplified DNA.

Whole Genome Amplification Directly from Archived TissueThe GenomePlex Tissue Whole Genome Amplification Kit (WGA5) contains all of the reagents necessary to amplify genomic DNA directly from formalin-fixed, paraffin-embedded (FFPE), frozen, RNAlater®-preserved or fresh tissue. The kit includes optimized reagents for tissue disruption and cell lysis eliminating the need for tedious organic extractions to remove excess paraffin or DNA purification prior to amplification. This rapid and straightforward method yields microgram quantities of genomic DNA from one milligram of tissue.

DNA isolated from archived tissue is often of poor quality making it unsuitable for most commercially available whole genome amplification kits that require high quality DNA as starting material. The flexibility of the GenomePlex technology enables the use of compromised starting material, such as DNA isolated from FFPE tissue, for whole genome amplification.

GenomePlex Flexibility Allows Amplification of Compromised Input DNAThe GenomePlex Tissue Whole Genome Amplification Kit enables the amplification of microgram quantities of DNA directly from 1 mg of FFPE tissue with minimal allele bias.

1 2 3 4 5 6 7

Figure 6: DNA was amplified directly from 1 mg of various formalin-fixed, paraffin-embedded human tissues with the GenomePlex Tissue WGA Kit (WGA5). DNA was purified with the GenElute™ PCR Clean-up Kit and analyzed on a 1% Agarose Gel. WGA yields were 5-6 micrograms for all samples.

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To view the Drug Discovery & Development and G&P webcast Advances in Genetic Analysis of Archived Samples, visit sigma.com/webcasts

1. Wide Range Marker

2. Uterus sample 1

3. Uterus sample 2

4. Gall Bladder

5. Colon

6. No Template control

7. Wide Range Marker

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Whole Genome Amplification from a Single Cell The GenomePlex Single Cell Whole Genome Amplification Kit (Cat. No. WGA4) is designed to amplify the genome of a single cell. This rapid and straightforward method provides million-fold amplification yielding microgram quantities of genomic DNA from a single cell. Traditional single cell whole genome amplification methods yield insufficient quantities with significantly biased representation. In contrast, the GenomePlex technology provides enhanced amplification efficiency by using WGA DNA polymerase.

2 kb

1 kb

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0.2 kb

Lane 1: Wide Range DNA Marker

Lane 2: Single Cell 2

Lane 5: Single Cell 5

Lane 4: Single Cell 4

Lane 3: Single Cell 3

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Human leukemia U937 cells were isolated using flow cytometric analysis and sorting (FACS), lysed, and amplified using the GenomePlex Single Cell Whole Genome Amplification Kit. The DNA was then purified with the GenElute™ PCR Cleanup Kit. An estimated million-fold amplification from the WGA pro-cess resulted in a final yield ranging from 5.4-6.2 mg. The Single Cell WGA Kit produces consistent yield and size range as visualized by a 1% agarose gel.

The Single Cell WGA kit includes all of the reagents necessary for cell lysis and subsequent whole genome amplification. Single cells can be isolated by fluorescence-activated cell sorting (FACS), laser capture microdissection (LCM), dilution, or any other applicable method. Single Cell WGA has been successfully applied to Comparative Genome Hybridization (CGH), STR analysis of amniocentesis samples, and genomic analysis of in vitro fertilized embryos.

Human Control Chromosome 3 Human Kidney Tumor Chromosome 3

DNA from normal and tumorigenic human kidney cells were amplified using the GenomePlex Single Cell WGA Kit. Amplified material was hybrid-ized to metaphase BAC arrays to determine chromosomal karyotype. Data that falls outside of the red line indicates chromsomal loss, while data that continues past the green line suggests chromosomal amplification. As expected the control sample demonstrated a balanced chromosomal copy number. Chromosome 3 for the amplified kidney tumor single cell displayed under representation as depicted by the red bar. These results match previ-ous microarray work using an amplification method that took three days. GenomePlex technology accurately amplifies genomic material down to single cell resolution. Data is courtesy of Dr. Michael Speicher from the Institute of Human Genetics, TU Munich.

To see Sigma’s complete line of Whole Genome Amplification products, visit our Web site at sigma.com/wga.

Ordering InformationCat. No. Product Name Quantity

WGA2 GenomePlex Complete Whole Genome Amplification (WGA) Kit 10 reactions 50 reactions

WGA4 GenomePlex Single Cell Whole Genome Amplification Kit 10 reactions 50 reactions

WGA3 GenomePlex WGA Reamplification Kit 50 reactions

WGA5 GenomePlex Tissue Whole Genome Amplification Kit 10 reactions 50 reactions

NA1020 GenElute PCR Clean-Up Kit 1 kit

G1N10 GenElute Mammalian Genomic DNA Miniprep Kit 1 kit

S4438 SYBR® Green JumpStart™ Taq ReadyMix™ 100 reactions 500 reactions

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SYBR® Green JumpStart Taq ReadyMixReal time detection in a complete reagent

SYBR Green JumpStart Taq ReadyMix for Quantitative PCR combines the advantages of a hot start enzyme with a ready-to-use mix, making it the ideal choice for high throughput, quantitative PCR. The ReadyMix includes the fluorescent dye SYBR Green I, JumpStart Taq DNA Polymerase, 99% pure deoxynucleotides and buffer in an optimized 23 concentrate.

SYBR Green JumpStart Taq ReadyMix is recommended for single product real time amplification experiments. It may also be used for evaluation of primer sequences prior to manufacture of fluorescent-labeled primers. Fluorescent-labeled primers are not recommended for use with SYBR Green I dye.

SYBR Green I, a commonly used fluorescent DNA binding dye, binds all double-stranded DNA and thus product accumulation is detected by measuring the increase in fluorescence throughout the cycle. SYBR Green I has an excitation and emission maxima of 494 nm and 521 nm, respectively.

JumpStart Taq Polymerase is an antibody inactivated hot start enzyme designed to minimize non-specific amplification while increasing target yield resulting in more accurate Ct values and improved standard curve for absolute sample quantitation.

To prepare a reaction, add 25 ml of ReadyMix to primers, template and water for a final reaction volume of 50 ml.

Sigma’s Reference Dye for Quantitative PCR is included with this ReadyMix for normalization of the reaction data. The dye has a maximum excitation of 586 nm, and a maximum emission of 605 nm. The instrument settings for ROX Reference Dye are suitable for the measurement of the Reference Dye for Quantitative PCR.

Ct Values for the Lambda Amplicon Using SYBR Green JumpStart Taq ReadyMix

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

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Ct values for the lambda amplicon using SYBR Green JumpStart Taq ReadyMix. Quantitative PCR (qPCR) was performed on pBac-2cp. Initial template copy number was 106 and was diluted 10-fold in subsequent wells. Threshold cycles (Ct) were determined using the ABI PRISM 7700 Sequence Detection software, and were found to be 15.304 (106), 18.848 (105), 22.883 (104), 26.208 (103), 29.821 (102), 33.398 (101), 37.038 (100), and 40 (0).

Components: SYBR Green JumpStart Taq ReadyMix Reference Dye for Quantitative PCR Separate Vial of 25 mM MgCl2 Included in S5193

Unit definition: One unit incorporates 10 nmol of total dNTPs into acid-precipitable DNA in 30 min at 74 °C

Concentration: 1.5 units per reaction (50 ml reaction volume) Storage: –20 °C Shipped in wet ice

Ordering InformationCat. No. Product Description Quantity

S4438 SYBR Green JumpStart 100 reactions Taq ReadyMix 500 reactions

S5193 SYBR Green JumpStart 100 reactions Taq ReadyMix 400 reactions without MgCl2

Qu

antitative PC

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Quantitative PCR Product Listing Cat. No. Product DescriptionSYBR-based S4438 SYBR Green JumpStart Taq ReadyMix

S1816 SYBR Green JumpStart Taq ReadyMix, Capillary Formulation

S5193 SYBR Green Jumpstart Taq ReadyMix without MgCl2S9194 SYBR Green ReadyMix for High Throughput Quantitative PCR

QR0100 SYBR Green Quantitative RT-PCR Kit

Probe-basedD7440 JumpStart Taq ReadyMix for Probe-based qPCR applications

D6442 JumpStart Taq ReadyMix for High Throughput Quantitative PCR

QR0200 Quantitative RT-PCR ReadyMix for Probe-based applications

PPD1 PCR Plate Detection Kit (sufficient for 480 detection reactions)

R4526 Reference Dye for Quantitative PCR

Quantitative PCR

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JumpStart™ Taq ReadyMix™ for High Throughput Quantitative PCR JumpStart Taq ReadyMix for High Throughput Quantitative PCR combines the advantages of a hot start enzyme with the convenience of an easy-to-use reaction mixture. The 23 concentrated ReadyMix contains JumpStart Taq DNA polymerase, 99% pure deoxynucleotides, reaction buffer, and an internal reference dye for ABI and other real time instrument applications. To set up the reaction simply add an equal volume of the ReadyMix to a 23 mixture of DNA template, primers and fluorescent probe.

n Internal reference dye incorporated into ReadyMix for quick set-up

n High throughput ReadyMix contains all necessary reagents except probe, primers and template DNA

n Hot Start PCR for more accurate Ct values and improved quantitation

Ordering InformationCat. No. Product Description Quantity

D6442 JumpStart Taq ReadyMix 400 reactions for High Throughput 2,000 reactions Quantitative PCR

SYBR® Green ReadyMix for High Throughput Quantitative PCR SYBR Green ReadyMix for High Throughput Quantitative PCR combines the performance enhancements of JumpStart Taq and SYBR Green I in an easy-to-use ReadyMix solution. The 23 concentrated ReadyMix contains the SYBR detection fluor, JumpStart Taq DNA polymerase, 99% pure deoxynucleotides, reaction buffer, and an internal reference dye for ABI and other real time instrument applications. To set up the reaction simply add an equal volume of the ReadyMix to a 23 mixture of DNA template and primers.

n SYBR Green I dye for nonspecific sequence detection in quantitative PCR

n High throughput ReadyMix contains all necessary reagents, except primers, and template DNA

n Hot Start PCR for more accurate Ct values and improved quantitation

Ordering InformationCat. No. Product Description Quantity

S9194 SYBR Green ReadyMix 400 reactions for High Throughput 2,000 reactions Quantitative PCR

Quantitative PCRQ

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1) Set up your PCR reaction with the SYBR Green JumpStart Taq ReadyMix. The mix contains enzyme, dNTPs, buffer and SYBR Green I dye. Just add your primers and template.

2) As the reaction progresses, double-stranded products are generated. The SYBR Green I dye intercalates into these products and begins to fluoresce.

3) When enough products have accumulated the fluorescence rises above background. This is called the threshold cycle or Ct. The Ct value is used to quantify the starting amount of template.

2 3

Proven!

Our Innovation, Your Research — Shaping the Future of Life Science

Advanced Quality. Reliable Performance. Superior Oligos. At Sigma-Genosys quality starts with high-quality raw materials, and ends with state-of-the-art analytical techniques like mass spectrometry analysis.

Our ISO 9001:2000 registered quality management system ensures:n Reproducible qualityn Consistent and quick deliveryn Traceability of raw materials from beginning to endn Reduced prices due to controlled costs

Every oligo sequence is guaranteed!

Sigma-Genosys manufactures high-quality custom products in 10 countries around the globe - Australia, Canada, France, Germany, India, Israel, Japan, Singapore, UK, and the USA.

ISO 9001:2000 registered: Canada, Germany, Israel, Japan, and the USA. ISO 14001:1996 registered: UK.

To learn more about our custom manufacturing capabilities for DNA, RNA, peptides or antisera, visit sigma.com/oligos.

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