382PR01

G-Biosciences, St Louis, MO. USA 1-800-628-7730 1-314-991-6034 technical@GBiosciences.com

A Geno Technology, Inc. (USA) brand name

1-Step RT-PCR Kit

INTRODUCTION The 1-Step RT-PCR Kit is designed for optimal convenience in carrying out highly sensitive and specific RT-PCR in a single tube. 1-Step RT-PCR is a variation on standard (two-step) RT-PCR in which all components of the RT and PCR are mixed in one tube prior to starting the reactions and in which RT and PCR are thus carried out sequentially in one tube. This approach offers tremendous convenience when applied to analysis of single targets from multiple samples of RNA. This approach also minimizes the possibility for introduction of contaminants into reactions between the RT and PCR steps, since the RT and PCR reactions are carried out sequentially, without opening of reaction tubes between steps. The kit is composed of high quality reagents. The AMV reverse transcriptase, from Avian Myeloblastosis Virus, is used for RT, and the optimized Taq DNA polymerase is used for PCR. A unique buffer, 10RT-PCR buffer suitable for both the RT and PCR steps and crucial for successful 1-Step RT-PCR, is included, along with dNTPs mixture and Ribonuclease Inhibitor. DEPC-treated water is also provided. Use of this kit and the accompanying protocol will ensure excellent results. ITEMS SUPPLIED Part. # 219A-B 003T-I 068R-A 210D-A 079R-B 025D-F Description AMV Reverse Transcriptase [25U/l] Taq DNA Polymerase [5U/l] RT-PCR Buffer [10X] dNTPs [10mM each] Ribonuclease Inhibitor [40U/l] DEPC-treated water Cat. # 786-813 Size 125U 125U 50l 25l 300U 0.5ml

STORAGE CONDITIONS The kit is shipped on blue ice. Upon arrival, store at -20C. This product is stable for 1 year at -20C. The AMV Reverse Transcriptase is supplied in 200mM potassium phosphate (pH7.2), 2mM DTT, 0.2% Triton X-100, 50% Glycerol. SOURCE AMV Reverse Transcriptase: Avian Myeloblastosis Virus Taq DNA Polymerase: The polymerase gene from Thermus aquaticus (strain YT1) is cloned and expressed in E. coli, then highly purified to produce Taq DNA polymerase.

UNIT DEFINITION AMV Reverse Transcriptase: One unit is defined as the amount of enzymes that incorporate 1nmol of dTTP into acidinsoluble precipitate in10 min using poly(A)+ RNA and oligo(dT) as the template/primer at 37C. Taq DNA Polymerase: One unit (U) of Taq polymerase is defined as the amount of enzyme needed to catalyze the incorporation of 10 nanomoles of deoxyribonucleotides into acid-insoluble material in 30 minutes at 70C using herring sperm DNA as a substrate.

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IMPORTANT INFORMATION Ensure the integrity and purity of your RNA. The quality of RNA is the key for first-strand cDNA synthesis. The integrity and purity of RNA can be inspected by the ratio of OD260/OD280 and agarose gel analysis. The common ratio of purified RNA is 1.8~2.0, if not, the RNA should be purified further. The ratio of eukaryotic RNA 28S/18S is about 2:1, if not, the RNA has been degraded. Avoid RNase contamination. All vessels, reagents and solutions must be sterile, and all procedures must be performed with gloves.

PROTOCOL 1. Add the following components into 0.2ml or 0.5ml thin-wall PCR tube: Reagent Total RNA Forward primer Reverse primer RT-PCR Buffer [10X] dNTPs [10mM each] AMV Reverse Transcriptase [25U/l] Taq DNA Polymerase [5U/l] Ribonuclease Inhibitor [40U/l] DEPC- treated water 2. 3. 4. Volume 0.5~1g 10~30pmol 10~30pmol 2l 1l 0.2l 1l 0.25l up to 20 l

Mix the solution and centrifuge briefly, then incubate for 30 min at 45C. Stop the reaction by incubating at 94C for 5 min. Immediately begin the PCR cycle. Use 30~40 cycles with the following recommend parameters: a. b. c. 94C for 30 seconds 60C for 45 seconds 72C for 1-3 minutes

5.

Finish the PCR amplification with an extension at 72C for 7 minutes.

TROUBLESHOOTING Issue Possible Cause Quality of template RNA was too low Concentration of RNA was too high Low yield of Reverse transcriptase inhibitor existed or cDNA reverse transcriptase was insufficient Reaction volume was too large RNA has been degraded Limited full length cDNA Poor reaction conditions Inhibited by RNA secondary structure

Suggestion See Important Information for RNA Purity & Integrity Assay RNA concentration and dilute. Use 0.5-1g. See Important Information for RNA Purity & Integrity Maximum volume should be 50l Ensure all equipment, pipettes and gloves are RNase free. Ensure Ribonuclease Inhibitor is in the reaction. Optimize the quantity of reverse transcriptase, DTT concentration (0.5-10mM) and reaction temperature (37-56C) Increase reaction temperature or use a random primer

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RELATED PRODUCTS 1. M-MLV Reverse Transcriptase (Cat. # 786-811). Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase is a RNA-dependent DNA polymerase. This enzyme is able to use RNA molecule as a template and synthesize single-strand DNA. 2. AMV Reverse Transcriptase (Cat. # 786-811). Synthesis of cDNA using RNA as template, RT-PCR, RNA and DNA sequencing. 3. First-Strand cDNA Synthesis Kit (Cat. # 786-812). A complete system for efficient first-strand cDNA synthesis from total RNA or mRNA. This kit contains oligo(dT)18 and random nonamer primers according to different experimental needs. The kit uses M-MLV Reverse Transcriptase. 4. 2-Step RT-PCR Kit (Cat. # 786-814). All the reagents needed to synthesize the first-strand cDNA followed by amplification of the cDNA product using PCR. Oligo(dT)18 and random nonamer primers (dN)9 are provided according to different experimental needs. The kit uses M-MLV Reverse Transcriptase.

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Last saved: 8/25/2011 CMH

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