MasterAmp™ High Fidelity RT-PCR Kit

MasterAmp™ High Fidelity RT-PCR Kitは、クローニング、シーケンス、発現解析などに使用できる正確な2本鎖cDNAを合成するキットです。

このキットは逆転写酵素にMMLV-RT PlusをまたDNA Polymeraseに MasterAmp™ TAQurate™ DNA PolymeraseのDNA Polymerase mixを採用しています。

また、キットには最適化された 2X RT-PCR Buffer PreMix (dNTPs, MgCl2, bufferを含む)と random primerと oligo(dT) primers及び betaine^*を含むMasterAmp™ PCR Enhancerが添付されます。MasterAmp PCR EnhancerはRT-PCRにおける特異性と収量を改善します。 MasterAmp™ 10X PCR Enhancerは個別に販売しています。

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.

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

• High-fidelity RT-PCR (i.e., low error rates, Figure 1).
• Amplification of full-length products from RNA templates up to ~6 kb.(Figure 2).
• Random and 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.

Protocols for One-Step or Two-Step Applications

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.

Contents:

• MMLV-RT Plus
• MasterAmp™ TAQurate™ DNA Polymerase Mix
• MasterAmp™ 2X RT-PCR PreMix (includes dNTPs)
• MasterAmp™ 10X PCR Enhancer
• Random Nonamer Primer
• Oligo (dT)18 Primer
• Control Template and Primer Mix
• Sterile Water

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