PI-31005,31016
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1. all reactions contain the same concentration of ROX In general ABI 7900 needs more ROX reference dye than ABI 7500 Thermal Cyclers such as iCycler iQ5 iCycler IQ MJ Opticon MJ Chromo 4 Rotor Gene 3000 Rotor Gene 6000 Rotor Gene Q or LightCycler 480 do not need ROX reference dye for normalization 3 Hydrolysis probes such as TaqMan probe cannot be used for melt curve analysis To take advantage of both probe specificity and melt curve capability we recommend a multiplex PCR reaction with AllGlo probes and EvaGreen dsDNA binding dye in one single tube see reference 2 Master Mix Selection Guide for Real Time Instruments Cat Product PCR Instrument 31005 Fast Probe Master Mix BioRad iCycler MyiQ MiQ 2 iQ 5 CFX 96 CFX 384 MJ Opticon Option2 Chromo4 MiniOpticon Qiagen Roto Gene Q Roto Gene3000 Roto Gene 6000 Eppendorf Mastercycler realplex Illumina Eco RealTime PCR System Cepheid SmartCyler Roche LightCycler 480 LightCycloer 2 0 31016 Fast Probe ABI 7500 7500 Fast 5700 7000 7300 Master Mix with 7700 7900 7900HT 7900HT Fast StepOne ROX StepOne plus Stratagene MX4000P MX3000P MX3005P AllGlo MAR JUP SAT URA NEP and PLU are trademarks of AlleLogic Biosciences Corporation AllGlo dyes and probes and their use are covered by US and international patents U S Patent No 7 667 024 TaqMan and Molecular Beacon is a registered trademark of Molecu2. Biotiu m 3159 Corporate place Hayward CA 94545 www biotium com Glowing Products for Science Last updated November 10 2010 Product Information Fast Probe Master Mix Catalog Number 31005 31005 1 31005 2 or 31005 T Unit Size 200 reactions cat 31005 500 reactions cat 31005 1 5 000 reactions cat 31005 2 or 100 reactions cat 31005 T trial size Components The product is 2X probe master mix that contains dNTP optimized buffer and stablizer including Tris and MgCl and Cheetah hot start Taq polymerase Cat Unit Size 31005 200 rxn 2 X 1 mL 31005 1 500 rxn 5 X 1 mL 31005 2 5 000 rxn 50 X 1 mL 31005 T 100 rxn 1 X 1 mL Fast Probe Master Mix with ROX Catalog Number 31016 31016 1 31016 2 or 31016 T Unit Size 200 reactions cat 31016 500 reactions cat 31016 1 5 000 reactions cat 31016 2 or 100 reactions cat 31016 T trial size Components The product is 2X probe master mix that contains dNTP optimized buffer and stablizer including Tris and MgCl Cheetah hot start Taq polymerase and ROX reference dye which may be required on ABI and certain other instruments See master mix selection guide for real time instruments below This product is suitable for all color probes qPCR detection except for ROX channel probe If you use ROX equivalent wavelength dye labeled probes URA as in AllGlo probe please choose Fast Probe Master Mix
3. Cat 31005 instead Cat Unit Size 31016 200 rxn 2 X 1 mL 31016 1 500 rxn 5 X 1 mL 31016 2 5 000 rxn 50 X 1 mL 31016 T 100 rxn 1 X 1 mL Storage and Handling Fast Probe Master Mix is shipped on blue ice and should be stored imme diately upon arrival at 20 C When stored in a constant temperature freezer at 20 C the kit is stable for at least 6 months from the date of receipt Before use thaw at room temperature and mix thouroghly by gentle vortexing After thawing the master mix should be kept on ice before use It can be refrozen for storage Product Description Fast Probe Master Mix is a ready to use hot start mix for quantitative real time analysis of DNA samples from various sources using probe based detection Although optimized for AllGlo probe system this master mix is also suitable for other probe systems such as hydrolysis probes such as TaqMan and Molecular Beacon The AllGlo probe is a simple fluorogenic probe without quencher or MGB AllGlo probes generate two light emitting labels per probe giving more intense fluorescence than traditional Taqman probes AllGlo probes is available in six colors MAR JUP SAT URA NEP and PLU which match with the absorption and emission wavelength of FAM HEX TAMRA ROX Cy5 and Quasar 670 respectively These six AllGlo dye labels are compatible with most real time PCR instruments and generally require no special calibr
4. ach 0 1 0 5 uM each primer the high the signal the reaction generates 2 ROX reference dye ROX reference dye is Probe x uL each 0 1 0 5 uM each not required for most of non ABI instruments and not supplied in the mix Cat 31005 For well to well fluorescence normalization in ABI and Template x uL See Helpful Tip 1 some other instrument platforms use the mix with ROX Cat 31016 which ROX reference dye is conveniently included Please refer to the ROX Optional See Helpful Tip 2 master mix selection guide in page 2 to decide which master mix is best suited for real time HO Add to 20 uL instruments 2 Cycling Protocol You may choose one of the following three protocols depending on the nature of your amplicon and instrument capability A Two step fast cycling protocol This cycling protocol should be applicable to hydrolysis probes when the amplification is short and the primer and probe T s are close to 60 C Number Helpful Tip of Cyles 3 Denaturation time The holding time for dena turation can be lower than 5 seconds including Cycling Step Temperature Holding Time EYA a as low as 0 second if you have a relatively short Efe ying acHyation a zimin 1 amplicon When the denaturation time is set to 0 second in the program it merely means that Denaturation 96 C 5 s See Helpful Tip 3 the temperature will ramp to 96 C and then will 40 immediately ramp d
5. ation For detailed probe design guidelines refer to AllGlo user s manual www allelogic com This specially formulated probe master mix offers strong signal and high sensitivity which make gene quantitation and viral load assessment possible even for the most challenging samples such as tissue blood or serum The wide range of reporter dye choices enable multi gene detection in a single tube and provide reliable results for multiplex applications such as SNP gene typing and pathogen detection Although the master mix is formulated for qPCR using a fast cycling protocol it is also compatible with qPCR using a regular cycling protocol see below for recommended cycling protocols of Two Step fast Univeral and Three Step regular ROX reference dye is not required for most of non ABI instruments and not supplied in the mix Cat 31005 For well to well fluorescence normalization in ABI and some other instrument platforms use the mix with ROX Cat 31016 which ROX reference dye is conveniently included Please refer to the master mix selection guide below to decide which master mix is best suited for real time instruments Another critical component of the master mix is Cheetah Taq our proprietary chemically modified hot start DNA Polymerase Unlike AmpliTaq Gold which is also a chemically modified Taq but takes 10 minutes or longer to activate Cheetah Taq is fully recovered in 2 minutes with high activity making it particular
6. ave an AT rich Enzyme activation 95 C 5 min 1 patch extension at 72 C should be avoided Denaturation 95 C 15s Annealing 50 60 C See Helpful Tip 4 30 s 40 Extension 72 C See Helpful Tip 5 30s 3 Data Acquisition Probe based fluorescence data acquisition depends on the chemistry used For AllGlo probe or TaqMan chemistry please refer to the following table for the excitation and emission wavelengths for each dye label AllGlo Probe Dye Spectral Properties AllGlo Dye Detection Channel Excitation Peak Emission Peak nm nm MAR FAM 495 520 JUP HEX JOE VIC 525 550 SAT TAMRA 555 580 URA ROX TEXAS RED 575 600 NEP Cy5 635 670 PLU Quasar 670 Alexa 680 685 720 a Biotium 4
7. c Homo Labeled Probe Technol ogy for Real Time qPCR 3rd International qPCR Symposium 26th 30th March 2007 in Freising Weihenstephan ISBN 13 978 3 00 020385 5 2 Xin X A closed tube real time PCR system for simultaneous pathogen detection and mutation scanning 2008 11th WPCCID PS3 078 3 Mao F Leung WY Xin X Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications BMC Biotechnol 2007 Nov 9 7 76 Additional Notes 1 Data analysis Absolute quantification is performed by plotting samples of unknown concentration to a standard curve from a dilution series of template DNA of known concentrations A standard curve is first generated by plotting Ct y axis threshold cycle against the logarithm of DNA amount x axis copy number A linear regression is used to calculate the amount DNA of unknown samples The slope of this line represents the efficiency of PCR reaction which should be 3 3 if the efficiency is 100 2 ROX reference dye Fast AllGlo Probe Master Mix with ROX Cat 31016 contains optimal ROX reference passive dye which is required on ABI and some other instruments If you use ROX equivalent wavelength dye labeled probes URA as in AllGlo probe please choose Fast Probe Master Mix Cat 31005 instead The concentration of ROX in a reaction will artificially affect the apparent read out of the fluorescence signals but it will not affect the relative Ct as long as
8. lar Probes Inc Practicing real time PCR may require a license from Roche or Applied Biosystems Inc Related Products EvaGreen dye 20X in H O Cat 31000 Cheetah Hotstart Taq DNA polymerase Cat 29050 Fast EvaGreen Master Mix Cat 31003 Fast EvaGreen Master Mix with Low ROX Cat 31014 Fast EvaGreen Master Mix with High ROX Ca t 31015 dNTP Set 100mM Cat 40052 dNTP Mix 25mM Cat 40053 dNTP Mix 10mM Cat 40054 PMA for selective detection of live pathogens by PCR Cat 40013 GelRed nucleic acid gel stain 10 000X in H O Cat 41003 GelGreen nucleic acid gel stain 10 000X in H O Cat 41005 a Biotium 2 PCR Protocols 1 Reaction Setup To prepare probe stock refer to the probe amount listed on the Oligonucleotide Specification Sheet For each 1 nmole of fluorescence labeled probe add 100 ul dH20 to make 10 uM stock which can be used as 20X if the final probe concentration is 500 nM Pipet reaction components into each well according to the table below Amount required for Final concentration Helpful Tips ee Pee 20 uL reaction 1 Amplicon length To maximize amplification efficiency with Fast Probe Master Mix the optimal amplicon length is 50 100 bp If longer amplicon 10 uL 1X is intended you may need to extend elongation Reaction component 2X Fast Probe Master Mi ge time For hydrolysis probes which require 5 exonuclease activity the close the probe to the Primers x uL e
9. ly suitable for fast PCR Cheetah Taq is completely inactive at room temperature and largely free of DNA contamination This makes Cheetah Taq superior to any antibody based hotstart Taq which is typically not completely inactive at room temperature and is prone to DNA contamination due to the nature of antibody production This kit is suitable for mRNA quantitation if a two step procedure is followed The first step involves converting the mRNA to cDNA by reverse transcription components not provided A portion of the synthesized cDNA can then be quantitated by using Fast Probe Master Mix in the second step To ensure optimal amplification efficiency the aliquot of the cDNA sample to be amplified should not exceed 10 of the volume of the PCR reaction For accurate quantitation of mRNA level a none RT control is recommended to eliminate the possibility of genomic DNA contamination a Biotium 1 For one step RT qPCR reverse transcriptase components not provided is required It is critical to have well characterized primer set that does not form primer dimer We recommend to titrate the amount of reverse transcriptase and the duration of the RT step If possible design primers to have Tm at 55 C run both RT step and extension step at 55 C For accurate quantitation of mRNA level a none RT control is recommended to eliminate the possibility of genomic DNA contamination References 1 Xin X and Mao F AllGlo Probe Highly Specifi
10. own with no delay Setting Annealing amp 60 C 30s the time to 5 s will ensures a more robust dena Extension turation for relatively long or high GC amplicons Instruments with fast ramping capability further add reliability to amplicon denaturation B Universal cycling protocol This traditional cycling protocol can be used on nearly all qPCR instruments The protocol may also benefit targets that are relatively difficult to amplify under fast cycling condition Cycling Step Temperature Holding Time umna Of Helpful Tip Cyles 4 Annealing amp Extension temperature The Tm s of primers and probe can be designed identical and as low as 50 C Accordingly the annealing Enzyme activa 95 C 5 min 1 amp extension temperature should be set to match tion the Tm of the primers This will benefit AT rich amplicons Denaturation 95 C 15s Annealing amp 50 60 C 60 s 40 Extension See Helpful Tip 4 a Biotium C Three step cycling protocol This cycling protocol can be used for probe systems like Molecular Beacons when Tm s of the probe is equal to or lower than 60 C An extra exten sion step at 72 C helps the polymerase to completely extend the product Cycling Step Temperature Behe bei Helpful Tips Time of Cyles 5 Extension temperature Taq extends most efficiently at 72 C However for AT rich amplicons tae o gt 70 AT or amplicons that h
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PI 3100531016