All you need for PCR. vwr.com. For PCR, RT-PCR, qpcr and qrt-pcr. A guide to instruments, reagents and consumables. I s s u e 1 - June 2006

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1 All you need for PCR A guide to instruments, reagents and consumables I s s u e 1 - June 2006 vwr.com Thermal cyclers including real time instruments plus all associated reagents Oligonucleotides and downstream processing of PCR products Consumable compatibility data with common use machines Adhesive and heat plate seals and accessory equipment For PCR, RT-PCR, qpcr and qrt-pcr

2 Polymerase Chain Reaction (PCR ) Introduction The intention of this guide is to provide a single document for all products needed to successfully perform PCR -based applications. For this purpose we have collected together appropriate products from our extensive range and put them into the guide. All suppliers presented in this catalogue hold the PCR license owned by Hoffman-La Roche Inc. Please note that the product selection may be subject to change. We have divided the guide into areas of different PCR -based applications, to make it easy to find what you need. In addition, there are sections on oligonucleotides, downstream processing of PCR products and PCR consumables, including an instrument/plate compatibility chart. Each section begins with a short introduction to the application, and is divided into subsections where the products are listed. The manufacturers are presented in alphabetical order. A small number of products have restricted availability in Europe. If there are any queries please contact your local VWR sales team. table of contents Instruments Thermal Cyclers... 3 Real-Time PCR Cyclers... 6 PCR Conventional Thermostable Polymerases... 7 Hot-Start Polymerases... 8 Hi-Fi Polymerases... 9 Buffers & dntps Master Mixes Pre-aliquoted Master Mixes PCR is a patented process of Hoffman-La Roche and requires a license. RT-PCR Reverse Transcriptases First-strand Synthesis Systems One-step RT-PCR Systems RT-Primers RT-Accessories q- and qrt-pcr q and qrt-pcr reaction mixtures presented according to compatibility with real time instruments Editor VWR International Ltd Customer Service Centre Hunter Boulevard Magna Park Lutterworth Leicestershire UNITED KINGDOM LE17 4XN PCR Consumables Compatibility Chart: Thermal Cyclers & Real-Time PCR Instruments / Plates... 24/25 Oligos Tubes & Strips Plates Plate Seals Copywriting VWR International Europe bvba Layout and typesetting The Graphic Design House, Portsmouth, UK Printing Bishops Printers, Portsmouth, UK No part of this publication may be reproduced or copied without prior permission by writing of VWR International Europe. Downstream Processing PCR Clean-Up Systems Molecular Weight Markers PCR Cloning Vectors Accessory Equipment Sealers Run copies Publication date: June 2006 VWR A l l y o u n e e d f o r P C R

3 q- and qrt-pcr Quantitative PCR (qpcr ), also referred to as real-time PCR, is a specific and extremely sensitive technique for quantitation of DNA template present in a sample of interest. If combined with reverse transcriptase it is called qrt-pcr and may be used to quantitate RNA. The great benefits of the technique are its sensitivity and specificity provided by the PCR reaction. The sensitivity is demonstrated by the fact that it is possible to quantitate the level of a specific mrna in a sample derived from a single cell. This vastly exceeds the sensitivity of alternative methods like Northern blot analysis and RNase protection assay. Quantitative PCR is based on real-time monitoring of PCR reactions. When the amplification of a template is observed throughout the PCR it is possible to predict the original amounts. Real-time monitoring is vital since the initial quantity of template in a PCR reaction is poorly reflected in measurements of the final product. The monitoring of PCR reactions are performed by detection of light emitted from reporter molecules (dyes or labelled probes). The basis for the system is the use of reporter molecules that signal only when bound to PCR products. In order to acheive specificity for the detection, two basic systems are mainly used, DNA binding dyes and probe based systems. DNA binding dyes bind to double stranded DNA (dsdna) irrespective of sequence. They emit fluorescence only when bound to dsdna and thus provide a measure of dsdna content in a reaction. These systems are not specific for a certain amplicon and are thus prone to produce false negatives. On the other hand, they are flexible and the same dye may be used to monitor any PCR reaction. An example of a DNA binding dye is SYBR Green. Probe based systems rely on labelled DNA oligos, designed to emit a signal only when bound to their respective sequence. The probes are basically one or two DNA molecules labelled with a regulator and a fluorophore. Regulation is acheived through the process of Fluorescence Resonance Energy Transfer (FRET). An exception is LUX primers that are negatively regulated by the secondary structure of the 3 end of the probe. Both negative and positive regulation are in use in different systems (see Fig. FRET). FRET occurs only over very small distances ( Å) which means that regulation may be performed by positioning of the regulator (donor or quencher) close to or away from the fluorophore. In the case of positive regulation a donor is attached to a primer whereas the reporter fluorophore is attached to another primer. The primers are designed to hybridise to sequences in close proximity to each other and thereby the donor is brought close to the fluorophore. The light from the instrument excites the donor which sends out a signal that excites the fluorophore. In response, the fluorophore emits a signal of a different wavelength that is detected by the instrument (Fig. FRET a). Negative regulation is achieved by using a quencher instead of an donor. The quencher is attached to a probe to which a fluorophore is attached as well. The quencher extinguishes the signal from the fluorophore. The result is that the instrument does not detect a signal as long as the fluorophore is kept close to the quencher. When the probe anneals to the template the quencher and the fluorophore are separated and the signal is detected. The method is far less laborious and does not involve use of potentially hazardous material like radiolabelled probes. Taken together, qpcr is more sensitive, faster and safer than its alternative methods. Fig. FRET a Schematic presentation of positive and negative regulation of light emission through FRET. Positive regulation is achieved when a donor fluorophore is situated adjacent to the reporter fluorophore. When the donor is excited it emits light in a wavelength that excites the acceptor fluorophore. That in turn, emits light of yet another wavelength detected by the instrument. Negative regulation functions in a similar manner but instead of a donor it utilises a quencher. In this case the fluorophore does not emit a signal when adjacent to the quencher and thus no light is detected by the instrument. VWR A l l y o u n e e d f o r P C R 17

4 This section of the guide is based on compatibility with real-time instruments. Commonly used real-time PCR instruments are presented as headings under which the compatible products for performing q- and qrt-pcr are presented. This means that you simply have to find the instrument you are going to use, and all the compatible products we supply are listed. Information regarding compatibility between instruments and plastics is available in the instruments/ plastics compatibility chart in the chapter for PCR consumables. ABgene ABgene provide mixes for performing qpcr and qrt-pcr with both probe based and SYBR Green I based detection of template. The mixes contain all components necessary except primers and template. The enzyme used for amplification is Thermo Start DNA Polymerase. For reverse transcription in the qrt-pcr mixes ABgene employ their unique blend of MmuLV and AMV RTases and a buffer designed for synergy with subsequent qpcr reactions. The unique QRTase Enhancer, that increase the efficiency of qpcr when performed using crudely purified or contaminated samples, provide mastermixes for performing qpcr for SYBR Green based detection as well as probe based detection. RealMasterMix is optimised for use with SYBR Green and single-plex, probe-based assays. The mix includes HotMaster Taq DNA Polymerase, a unique buffer system with stabilizers and dutp to minimize primer-dimer formation and s self-adjusting Mg 2 + system. Included is also ROX for passive reference dye is also included. Absolute qrt-pcr mixes are based on a balanced buffer to maximise performance of both the reverse transcription and DNA polymerase enzymes. In the SYBR Green mixes, the dye is also included in the buffer at an optimum concentration to facilitate detection of low abundance templates. N.B. dutp mixes are available. However, ABgene do not recommend the use of dutp as it is not incorporated as efficiently as dttp. If required, these mixes should only be used with heat labile UNG and at reverse transcription steps above 50 C. normalisation. RealMasterMix Probe is designed for optimal performance in probe based real-time PCR assays (e.g. TaqMan, molecular beacons). The mixes contain all systems included in the RealMasterMix with the exception that ROX is optional. Both types of mixes contain all components for qpcr except primers and template in one tube and are excellent for accurate real-time quantification with the detection system for which they are optimised. ABI Prism ABsolute QPCR ROX Mix 200 x 50 µl rxns ABsolute QPCR ROX & dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR ROX Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR ROX & dutp Mix 200 x 50 µl rxns ABsolute 2-Step QRT-PCR ROX Kit 200 x 50 µl rxns ABsolute 2-Step QRT-PCR ROX & dutp Kit 200 x 50 µl rxns ABsolute QPCR low ROX MIX 100 x 50 µl rxns ABsolute QPCR SYBR Green ROX Mix 200 x 50 µl rxns ABsolute QPCR SYBR Green ROX & dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green ROX Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green ROX & dutp Mix 200 x 50 µl rxns ABsolute 2-Step QRT-PCR SYBR Green ROX Kit 200 x 50 µl rxns ABsolute 2-Step QRT-PCR SYBR Green ROX & dutp Kit 200 x 50 µl rxns ABsolute QPCR SYBR Green Low ROX MIX 100 x 50 µl rxns VWR A l l y o u n e e d f o r P C R

5 For more information on these products visit our website Platinum SYBR Green qpcr SuperMixUDG w/rox 500 rxns SuperScript III Platinum SYBR Green OneStep 100 rxns qpcr Kit w/rox SuperScript III Platinum SYBR Green TwoStep 500 rxns qrtpcr Kit w/rox BioRad Icycler ABsolute QPCR Mix 200 x 50 µl rxns ABsolute QPCR dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR Mix 100 x 50 µl rxns ABsolute MAX QRT-PCR dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR dutp Kit 200 x 50 µl rxn ABsolute QPCR SYBR Green Fluorescein Mix 200 x 50 µl rxns ABsolute QPCR SYBR Green Fluorescein dutp Mix 200 x 50 µl rxns ABsolute QRT-PCR SYBR Green Fluorescein Mix 200 x 50 µl rxns ABsolute QRT-PCR SYBR Green Fluorescein dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR SYBR Green Fluorescein Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR SYBR Green Fluorescein & dutp Kit 200 x 50 µl rxn Platinum SYBR Green qpcr SuperMix-UDG 500 rxns SuperScript III Platinum SYBR Green One-Step qrt-pcr Kit 500 rxns SuperScript Platinum SYBR Green Two-Step qrt-pcr Kit with BSA 480 rxns SuperScript III Platinum CellsDirect Two-Step qrt-pcr Kit with SYBR Green 500 rxns VWR A l l y o u n e e d f o r P C R 19

6 Mastercycler ep Realplex No reagents have been specifically validated for use with this equipment from. However the system is compatible with most detection chemistries available. Cephid SmartCycler ABsolute QPCR Mix 200 x 50 µl rxns ABsolute QPCR dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR dutp Kit 200 x 50 µl rxn ABsolute QPCR SYBR Green Mix 200 x 50 µl rxns ABsolute QPCR SYBR Green dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR SYBR Green Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR SYBR Green dutp Kit 200 x 50 µl rxn The Cephid SmartCycler use specific consumables that require increased concentrations of BSA for optimal assay performance. It is feasible to use the RealMaster Probe kits with this platform; however, optimisation work will be required. 20 VWR A l l y o u n e e d f o r P C R

7 For more information on these products visit our website Corbett Rotor-Gene ABsolute QPCR Mix 200 x 50 µl rxns ABsolute QPCR dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR dutp Kit 200 x 50 µl rxn MJ Opticon ABsolute QPCR Mix 200 x 50 µl rxns ABsolute QPCR dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR dutp Kit 200 x 50 µl rxn ABsolute QPCR SYBR Green Mix 200 x 50 µl rxns ABsolute QPCR SYBR Green dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR SYBR Green Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR SYBR Green dutp Kit 200 x 50 µl rxn VWR A l l y o u n e e d f o r P C R 21

8 Roche LightCycler ABsolute QPCR Capillary Mix 250 x 20 µl rxn ABsolute QPCR Capillary dutp Mix 250 x 20 µl rxn ABsolute QRT-PCR Capillary Mix 250 x 20 µl rxn ABsolute QRT-PCR Capillary dutp Mix 250 x 20 µl rxn ABsolute 2-Step QRT-PCR Capillary Mix 500 x 20 µl rxn ABsolute 2-Step QRT-PCR Capillary dutp Mix 500 x 20 µl rxn ABsolute QPCR SYBR Green Capillary Mix 250 x 20 µl rxn ABsolute QPCR SYBR Green Capillary dutp Mix 250 x 20 µl rxn ABsolute QRT-PCR SYBR Green Capillary Mix 250 x 20 µl rxn ABsolute QRT-PCR SYBR Green Capillary dutp Mix 250 x 20 µl rxn ABsolute 2-Step QRT-PCR SYBR Green Capillary Mix 500 x 20 µl rxn ABsolute 2-Step QRT-PCR SYBR Green Capillary dutp Mix 500 x 20 µl rxn Roche Light-Cycler use specific consumables that require increased concentrations of BSA for optimal assay performance. It is feasible to use the RealMaster Probe kits with this platform; however, optimisation work will be required. Stratagene MX3000P/MX4000 ABsolute QPCR Mix plus ROX Vial 200 x 50 µl rxns ABsolute QPCR dutp Mix plus ROX Vial 200 x 50 µl rxns ABsolute MAX QRT-PCR Mix plus ROX Vial 200 x 50 µl rxns ABsolute MAX QRT-PCR dutp Mix plus ROX Vial 200 x 50 µl rxns ABsolute 2-Step QRT-PCR Mix plus ROX Vial 200 x 50 µl rxns ABsolute 2-Step QRT-PCR dutp Mix plus ROX Vial 200 x 50 µl rxns ABsolute QPCR SYBR Green Mix plus ROX Vial 200 x 50 µl rxns ABsolute QPCR SYBR Green dutp Mix plus ROX Vial 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green Mix plus ROX Vial 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green dutp 200 x 50 µl rxns Mix plus ROX Vial ABsolute 2-Step QRT-PCR SYBR Green Mix plus ROX Vial 200 x 50 µl rxns ABsolute 2-Step QRT-PCR SYBR Green dutp Mix 200 x 50 µl rxns plus ROX Vial 22 VWR A l l y o u n e e d f o r P C R

9 For more information on these products visit our website RealMaster Mix Probe 200 rxns RealMaster Mix ProbeRox 200 rxns Techne Quantica ABsolute QPCR Mix 200 x 50 µl rxns ABsolute QPCR dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR dutp Kit 200 x 50 µl rxn ABsolute QPCR SYBR Green Mix 200 x 50 µl rxns ABsolute QPCR SYBR Green dutp Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green Mix 200 x 50 µl rxns ABsolute MAX QRT-PCR SYBR Green dutp Mix 200 x 50 µl rxns ABsolute MAX 2-Step QRT-PCR SYBR Green Kit 200 x 50 µl rxn ABsolute MAX 2-Step QRT-PCR SYBR Green dutp Kit 200 x 50 µl rxn VWR A l l y o u n e e d f o r P C R 23

10 Go to for the latest news, special offers and full specifications of our complete product range The Market Source for Life Science Everything you need to succeed v w r b i o m a r k b e. v w r. c o m Austria VWR International GmbH Graumanngasse 7 A Wien Tel.: Fax: info@at.vwr.com Belgium VWR International bvba/sprl Haasrode Researchpark Zone 3 Geldenaaksebaan 464 B Leuven Tel.: Fax: customerservice@be.vwr.com Denmark VWR International ApS Roskildevej 16 DK Albertslund Tel.: Fax: info@dk.vwr.com Finland VWR International Oy Pihatörmä 1 C 1 FI Espoo Tel.: Fax: info@fi.vwr.com France VWR International S.A.S. Le Périgares Bâtiment B 201, rue Carnot F Fontenay-sous-Bois cedex Tel.: (0,15 EUR TTC/min) Fax: (0,15 EUR TTC/min) info@fr.vwr.com Germany VWR International GmbH Hilpertstrasse 20a D Darmstadt Tel.: Fax: info@de.vwr.com Ireland AGB Scientific Ltd - A VWR International Company Orion Business Campus Northwest Business Park Ballycoolin Dublin 15 Tel.: Fax: info@agb.ie Italy VWR International s.r.l. Via Stephenson 94 I Milano (MI) Tel.: Fax: info@it.vwr.com The Netherlands VWR International B.V. Postbus 8198 NL AD Amsterdam Tel.: +31 (0) Fax: +31 (0) info@nl.vwr.com Northern Ireland AGB Scientific Apparatus Ltd A VWR International Company A10 Harbour Court, 7 Heron Rd Sydenham Business Park Belfast BT3 9HB Tel.: Fax: info@agbscientific.co.uk Norway VWR International AS Kakkelovnskroken 1 P.B. 45, Kalbakken NO Oslo Tel.: Fax: info@no.vwr.com Portugal VWR International Material de Laboratorio, Lda. Portugal Rua Alfredo da Silva 3-C P Lisboa Tel.: /1/2/3 Fax: /9 info@pt.vwr.com Spain VWR International Eurolab S.L. Apartado 48 E Mollet del Vallés - Barcelona Tel.: Fax: info@es.vwr.com Sweden VWR International AB Fagerstagatan 18a SE Stockholm Tel.: Fax: info@se.vwr.com Switzerland VWR International AG Lerzenstrasse 16/18 CH Dietikon Tel.: Fax: info@ch.vwr.com UK VWR International Ltd Customer Service Centre Hunter Boulevard Magna Park Leicestershire LE17 4XN Tel.: Fax: uksales@uk.vwr.com ZPROVWRB2001-EN/EU