Contents > PCR & other Amplification Methods > MasterAmp™ High Fidelity RT-PCR Kit

MasterAmp™ High Fidelity RT-PCR Kit

Supplier: EPICENTRE® Biotechnologies

MasterAmp™ High Fidelity RT-PCR Kit

Catalogue number

Pack size

RF910100

100 Reactions

RF91025

25 Reactions

Covered by issued and pending patents.

* Epicentre's® PCR products are sold under licensing arrangements with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc., and Applied Biosystems.
The products containing a thermostable DNA polymerase are accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) and RT-PCR for life science research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorised thermal cycler.

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Last modified: 2008-07-15 14:57:15

Cells are lysed, and proteins precipitated. The DNA is alcohol precipitated.

Kit specifications
Kit	Yield
Non-spin	95%
300Bl-30ml blood	90%

The MasterAmp™ High Fidelity RT-PCR Kit is ideal for synthesis of accurate double-stranded cDNA products that will be used for cloning, sequencing, expression, or transcription analysis. The kit uses MMLV-RT Plus, a reverse transcriptase with an unsurpassed accuracy and reliability, and MasterAmp™ TAQurate™ DNA Polymerase, a high fidelity PCR enzyme mix.

The kit also contains an optimised 2X RT-PCR Buffer PreMix (containing dNTPs, MgCl2, and buffer), random and oligo dT primers, and the MasterAmp™ PCR Enhancer (with betaine)*. The PCR enhancer can substantially improve the yield and specificity of RT-PCR amplification. For more information about the MasterAmp 10X PCR Enhancer.

Figure 1. Sequence analysis of an RT-PCR product generated using MasterAmp™ High Fidelity RT-PCR Kit. The sequence of full-length (850 bp) rabbit tissue factor cDNA clone from rabbit brain mRNA was found to be error-free.

Figure 2. The MasterAmp™ High Fidelity RT-PCR Kit amplifies full-length products from RNA templates up to ~6 kb. RT-PCR products from Tobacco Mosaic Virus RNA with primers that amplify a 5.5-kb region of the viral RNA. Lane 1, kb ladder; Lane 2, RT-PCR using the MasterAmp one-step protocol; Lane 3, RT-PCR using MMLV-RT and Taq DNA polymerase with a standard two-step procedure; Lane 4, RT-PCR using Tth DNA polymerase.

Last modified: 2008-05-14 16:46:46

Applications

Highly accurate RT-PCR amplification of RNA templates for cloning, sequencing, expression, transcription analysis
All purpose RT-PCR in which full length amplification and high accuracy are essential
Multiplex RT-PCR

Benefits

Highest fidelity RT-PCR (i.e., low error rates)
Amplification of full-length products from long RNA templates
Random/oligo dT primers included for non-specific first strand cDNA synthesis
Unique PreMix provides convenience, reducing pipetting steps and chances of contamination
Uses MasterAmp™ PCR Enhancer Technology* for better amplification of high G+C or other difficult templates

ONE-STEP RT-PCR The one-step procedure performs first-strand cDNA synthesis and PCR in one tube using RNA specific primers.
STANDARD TWO-STEP RT-PCR In the two-step procedure, first-strand synthesis is performed using oligo(dT) or random primers. A small amount of cDNA is then used in subsequent PCR.
MODIFIED TWO-STEP RT-PCR In the modified two-step procedure, specific PCR primers and enzymes are added directly to the first-strand cDNA synthesis reaction for amplification by PCR.

Figure 3. Amplification of a variety of RNA templates using a one-step protocol with MasterAmp™ High Fidelity RT-PCR Kit. Lane 1, 100 bp ladder; Lane 2, human lung β-actin; Lane 3, human lung GAPDH; Lane 4, region of TMV; Lane 5, E. coli 16S rRNA; Lane 6, human CGα; Lane 7, human heart β-actin; Lane 8, region of TMV.

Figure 4. The modified two-step protocol using the MasterAmp™ High Fidelity RT-PCR Kit to amplify RNA reverse transcribed with non-specific primers. Total cellular RNA purified from human lung tissue was reverse transcribed with either message-specific (Lanes 2 and 5), random hexamer (Lanes 3 and 6), or oligo d(T)₁₈ (Lanes 4 and 7) primers prior to PCR with message-specific primers for β-actin or GAPDH.