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1Promega
Protocols & applications guide Chapter Updates! The PCR Applications chapter contains protocols and reference information on PCR, RT-PCR, hot-start PCR and qPCR. The updated Transfection chapter includes a summary of factors influencing transfection efficiency, useful tips on optimizing transfection experiments and sample transfection protocols.
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Scientific Posters
Introducing GoTaq® qPCR Master Mix: The Bright Choice for Dye-Based qPCR
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 Corporation, Madison, WI 2Promega Biosciences, LLC., San Luis Obispo, CA
1. Abstract 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
GoTaq® qPCR Master Mix
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. 2. Spectral properties of GoTaq® qPCR dye and SYBR® Green I
Company A Fast master mix
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.
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. 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.
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.
SYBR® Green I Promega Dye
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 May require addition of fluorescein as reference dye.** 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. 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.
Introducing GoTaq® qPCR Master Mix: The Bright Choice for Dye-Based qPCR
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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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A novel automated method to purify DNA and RNA from viruses: The Maxwell® 16 Viral Total Nucleic Acid Purification System
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New Interactive Brochure! Explore the advantages of our new GoScript™ Reverse Transcriptase product through a new interactive brochure.
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Educational Resources
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NEW! Science Writing Tips and Tricks is a series of articles about writing, with emphasis on science writing. These articles provide information about basic grammar and mechanics, the business of getting published as well as more advanced writing topics. Some topics include:
tips and tricks “Science Writing Tips and Tricks”
Accepted Without Revision suggestions about publishing your work in a peer-reviewed journal. Do the Math common errors to look for when writing technical material that includes calculations or statistics. The Notorious Not-A-Verb List some of the nouns that are often misused as verbs in writing. Use Words That You Understand the importance of using language precisely to communicate your message. Read these articles – and many more at Promega Science Writing page.
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Writing Tips
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