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1. Abstract 2. Spectral properties of GoTaq® qPCR dye and SYBR® Green I
SYBR® Green I Promega Dye
NEW PUBHUB ARTICLES! Using GoTaq qPCR Master Mix to Monitor Stem Cell Differentiation Quick and Easy Isolation of Genomic DNA from Drosophila Using the Maxwell® 16 Instrument
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Katharine Driftmier Miller Hoffmann1, Karen L. Reece1, Cesear Corona2, Thomas A. Kirkland2, H. Tetsuo Uyeda2, Stephen J. Dwight2, Mark G. McDougal2 and Douglas R. Storts1 1Promega Corporation, Madison, WI 2Promega Biosciences, LLC., San Luis Obispo, CA
4. GoTaq® qPCR shows earlier Cq than leading competitors 7. Excellent amplification efficiency and accurate quantification over a broad dynamic range 10. GoTaq® qPCR Master Mix is compatible with FAST cycling parameters
Company A Fast master mix GoTaq® qPCR Master Mix
Introducing GoTaq® qPCR Master Mix: The Bright Choice for Dye-Based qPCR
SCIENTIFIC POSTERS Monitoring Differential Expression in Stem Cells
GoTaq® qPCR Master Mix introduces a new, proprietary dsDNAbinding dye that can be used at a higher concentration than SYBR® Green I because it is less inhibitory in an amplification reaction. The dye concentration in the master mix is optimized to produce significantly brighter fluorescence during qPCR than master mixes containing SYBR® Green I. Excitation and emission of the dye are similar to those of SYBR® Green I, so it is compatible with commonly available instrumentation platforms. GoTaq® qPCR Master Mix is a 2X master mix composed of an optimized buffer formulation complete with dNTPs and MgCl2 and features GoTaq® Hot Start. The master mix comes premixed with a low level of CXR reference dye, which is identical to ROX™ reference dye. The master mix is compatible with existing experimental designs and requires addition of only DNA template, target-specific primers and water.
The GAPDH gene was amplified from 1ng of human genomic using GoTaq® qPCR Master Mix and six other commercially available dye-based master mixes using manufacturer protocols.
* Master Mixes were supplied without ROX reference dye. ROX was provided in the kit and added as recommended by the protocol.
Amplification of kanamycin from plasmid DNA over 8 logs orders. Inset blue squares represent 10-fold dilutions of plasmid DNA from 10 copies to 1 x 108 copies. Black crosses show accurate quantification of 10-fold dilutions from 30 to 30 x 107 copies.
GAPDH amplified from 0.01 – 10 ng human gDNA with GoTaq® qPCR Master Mix and Company A Fast master mix, using the ABI 7500 FAST default cycling parameters: Activation: 95°C for 20 sec. 40 cycles: Denaturation: 95°C for 3 sec Anneal/Extend: 60°C for 30 sec. Data shows GoTaq® qPCR Master Mix is compatible with FAST cycling parameters as is.
5. GoTaq® qPCR shows significantly higher fluorescence and earlier Cq at all template levels
8. Exceptional reproducibility across a 96-well plate
11. Instrument Compatibility
Use GoTaq ® qPCR Master Mix directly.
200 ng/µL dsDNA 50 ng/µL dsDNA
Applied Biosystems 7500 and 7500 Fast Real Time PCR Systems Stratagene Mx3005P® Quantitative PCR Systems Roche LightCycler® 480 BioRad Chromo4™ Real-Time Detector Eppendorf Mastercycler® ep realplex4 and realplex4 S* Require addition of 100X CXR. Reference Dye to 1X per reaction. Applied Biosystems 7000 Sequence Detection System Applied Biosystems 7300 Real-Time PCR System Applied Biosystems 7700 Sequence Detection System Applied Biosystems 7900HT Real-Time PCR System Applied Biosystems StepOne® and StepOne® Plus Real-Time PCR Systems Performance comparison of GoTaq® qPCR Master Mix and Company A's master mix. GAPDH was amplified from tenfold serial dilutions (0.01–100 ng) of human genomic DNA. Inset shows comparison of dissociation profiles for both master mixes. GoTaq® qPCR shows significantly higher fluorescence and earlier Cq at all template levels. May require addition of fluorescein as reference dye.** GoTaq® qPCR shows very tight distribution. Variability of normalized fluorescence intensity (dRn) of GoTaq qPCR Master Mix and Company A. GAPDH was amplified from 1ng of human gDNA, n=96. MyiQ™ System iQ™5 Real-Time PCR Detection System
*Users must use clear-well plates with these instruments. **Users should run a test reaction using their experimental target to determine if it is necessary to add fluorescein to the master mix.
buffer
Fluorescence spectra of SYBR® Green I and the Promega proprietary dye in GoTaq® qPCR Master Mix. Fluorescence emission spectra were collected for both SYBR® Green I and the proprietary Promega in the absence and in the presence of different concentrations of dsDNA.
3. GoTaq® qPCR Master Mix provides high sensitivity in detecting a single copy gene
6. GoTaq® qPCR shows brighter fluorescent signal than leading competitors.
9. GoTaq® qPCR is stable after 24 hours at room temperature
12. Summary
24 hrs @ 22°C
• Early Cq and detection of low copy targets. • Enhanced stability for automated setup. • Direct substitute for SYBR® Green I products. • The robust, reliable performance of GoTaq® Hot Start. • The Promega PCR Performance Guarantee.
The GAPDH gene was amplified and detected from 10-fold serial dilutions (0.01ng to 100ng) of human genomic DNA. Inset shows the standard curve for the various dilutions (Slope=-3.2; R2=0.995).
GAPDH was amplified from 1ng of human gDNA using GoTaq® qPCR Master Mix and six other commercially available dye-based master mixes using manufacturer protocols.
The reaction is stable after 24 hours at room temperature making the product ideal for automated instruments. Human dystrophin gene was amplified with GoTaq® qPCR Master Mix after 24 hours at room temperature. Expected specific melt curve peak is at 81°C.
*Master Mixes IP and IE were supplied without ROX reference dye. ROX was provided in the kit and added to the master mix as recommended by the protocol.
© 2009 Promega Corporation. All Rights Reserved. GoTaq is a registered trademark of Promega Corporation. FAM and ROX are trademarks of Applera Corporation. SYBR is a registered trademark of Molecular Probes, Inc. Mx3005P is a registered trademark of Stratagene. LightCycler is a registered trademark of Roche Molecular Systems, Inc. Chromo4 is a trademark of Bio-Rad Laboratories, Inc. Mastercycler is a registered trademark of Eppendorf, Inc. StepOne is a trademark of Applera Corporation. MyiQ and iQ are trademarks of Bio-Rad Laboratories, Inc. Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.
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HaloTag® Technology for Protein Expression, Solubilization and Purification
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The Detecting Microbial Contamination Unit supplements courses that introduce students to basic microbiology. The lecture and laboratory introduce students to ATP- and culture-based methods for detecting microbes.
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TEACHING RESOURCES
FUGENE® HD TRANSFECTION OPTIMIZATION WORKSHEET Simply input data from a reporter gene assay and cell viability assay, and this worksheet will calculate averages and standard deviations of replicates, and provide a graph to help determine optimal transfection conditions.
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