SuperScript?IIIOneStepRTPCRSystemwithPlatinum?TaqHighFidelity

實驗概要

The  SuperScript? III One-Step RT-PCR System with Platinum? Taq  High  Fidelity is designed for sensitive, high-fidelity end-point detection  and analysis of RNA molecules by RT-PCR. Using this convenient one-step  formulation, you can perform both cDNA synthesis and PCR amplification  in a single tube using gene-specific primers and target RNAs from either  total RNA or mRNA. The system uses a mixture of SuperScript? III  Reverse Transcriptase and Platinum? Taq  DNA Polymerase High Fidelity  for enhanced RT-PCR yields and fidelity, as well as the detection of  longer templates. The system can detect a wide range of RNA targets from  300 bp to 10 kb, and is compatible with multiplex applications. The  amount of starting material can range from 1 pg to 1 μg of total RNA.

實驗原理

The  system consists of two major components: SuperScript? III RT/  Platinum? Taq High Fidelity Enzyme Mix and 2X Reaction Mix. SuperScript?  III Reverse Transcriptase is a version of M-MLV RT that has been  engineered to reduce RNase H activity and provide increased thermal  stability.  The enzyme can synthesize cDNA at a temperature range of  45–60°C, providing increased specificity, higher yields of cDNA, and  more full-length product than other reverse transcriptases. Because  SuperScript? III RT is not significantly inhibited by ribosomal and  transfer RNA, it can be used to synthesize cDNA from total RNA.

Platinum? Taq   DNA Polymerase High Fidelity is an enzyme mixture composed of  recombinant Taq  DNA polymerase, Pyrococcus species GB-D polymerase, and  Platinum? Taq  antibodies, which block polymerase activity at ambient  temperatures. The antibodies are denatured and polymerase activity is  restored during the denaturation step in PCR cycling at 94° C, providing  an automatic “hot start” in PCR and increasing sensitivity,  specificity, and yield. Pyrococcus species GB-D polymerase is a  proofreading enzyme that possesses a 3’ to 5’ exonuclease activity.  Mixture of this enzyme with Taq  DNA polymerase results in a six-fold  increase in fidelity over Taq  DNA polymerase alone and allows  amplification of simple and complex DNA templates over a large range of  target sizes.

The  2X Reaction Mix included in the kit consists of a proprietary buffer  system that has been optimized for reverse transcription and PCR, and  includes Mg2 , deoxyribonucleotide triphosphates (dNTPs), and  stabilizers. A tube of 5 mM MgSO4 is included in the kit for further  optimization of the Mg2  concentration. Sufficient reagents are provided  for 25 or 100 amplification reactions of 50 μl each.

Note:   This  kit has been optimized for end-point RT-PCR. For quantitative real-time  RT-PCR, use the SuperScript? III Platinum? One-Step Quantitative RT-PCR  System.

主要試劑

主要設備

GeneAmp?PCR System 9600 and 2400

DNA Engine? PTC-200

實驗材料

RNA

實驗步驟

1. Program the thermal cycler so that cDNA synthesis is followed immediately with PCR amplification automatically.

2. Add  the following to a 0.2-ml, nuclease-free, thin-walled PCR tube on ice.  For multiple reactions, you can prepare a master mix to minimize reagent  loss and enable accurate pipetting.

3. Gently  mix and make sure that all the components are at the bottom of the  amplification tube. Centrifuge briefly if needed. Depending on the  thermal cycler used, overlay with silicone oil if necessary.

4. Place the reaction in the preheated thermal cycler programmed as described above. Collect the data and analyze the results.

注意事項

1. RNA Quality

1)            This kit has been optimized for use with 1 pg to 1 μg of total RNA.

2)            High quality intact RNA is essential for successful full-length cDNA synthesis.

3)            For low copy-number genes or longer targets, use more starting material (>10 ng total RNA).

4)            RNA should be devoid of any RNase contamination and aseptic conditions should be maintained.

5)            We  recommend the PureLink? Micro-to-Midi Total RNA Purification System or  TRIzol? Reagent for isolation of total RNA. See the Materials section  for ordering information. Oligo(dT) selection for poly(A)  RNA is  typically not necessary, although it may improve the yield of specific  cDNAs.

2. Primer Design

6)            We  recommend using gene specific primers (GSPs). We do not recommend using  oligo(dT) or random primers, as they can generate nonspecific products  in the one-step procedure and the amount of RT-PCR product may be  reduced.

7)            A  final primer concentration of 0.2 μM for each primer is generally  optimal. However, for best results, we recommend performing a primer  titration of 0.15-0.5 μM.

8)            Design  primers that anneal to the mRNA sequence in exons on both sides of an  intron or exon/exon boundary, to allow differentiation between the  amplified cDNA and potential contaminating genomic DNA.

9)            Primers should not be self-complementary or complementary to each other at the 3′ ends.

3. Magnesium and dNTP Concentration

10)        MgSO4 is  included in the 2X Reaction Mix at a final concentration of 1.2 mM,  which works well for most targets. If needed, the magnesium  concentration can further be optimized (usually between 1.2–2 mM) with  the 5-mM MgSO4 provided in the kit.

11)        dNTPs are included in the 2X Reaction Mix at a final concentration of 200 μM, which is optimal for most reactions.

4. Reaction Setup

12)        Program  the thermal cycler before setting up the reaction. The thermal cycler  should be preheated to 45–60° C, depending on the temperature selected  for cDNA synthesis.

13)        For difficult or high GC-content templates, use a 55–60° C cDNA synthesis temperature.

14)        Keep  all components, reaction mixes, and samples on ice. After preparation  of the samples, transfer them to the preheated thermal cycler and  immediately start the RT-PCR program.

15)        Efficient  cDNA synthesis can be accomplished in a 15–30-minute incubation at  45–60° C. For small targets, an incubation time of 5 minutes may be  sufficient.

16)        SuperScript? III RT is inactivated, Platinum^? Taq DNA Polymerase High Fidelity is reactivated, and the RNA/cDNA hybrid is denatured during the 2-minute incubation at 94° C.

17)        The annealing temperature should be 10° C below the melting temperature of the primers used.

18)        The extension time varies with the size of the amplicon (approximately 1 minute per 1 kb of amplicon).

19)        For all targets up to 10 kb, 1 μl of SuperScript? III RT/ Platinum^? Taq High Fidelity Enzyme Mix is sufficient.

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