Super One-step RT-PCR Kit for Virus Detection

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1 Super One-step RT-PCR Kit for Virus Detection For fast and sensitive one-step RT-PCR with highly specificity

2 SD Super One-step RT-PCR Kit Kit Contents Contents Cat. no. SD103 SD µl x 50 rxn 5 One-step RT-PCR Buffer 550 μl One-step Enzyme Mix 220 μl RNase-free ddh 2 O 1 ml 2 Handbook 1 Storage Super One-step RT-PCR Kit for Virus Detection should be stored at -20 C. Introduction Super One-step RT-PCR Kit provides a convenient format for highly sensitive and specific RT-PCR. The kit combines first-strand cdna synthesis and PCR amplification in the same tube. The One-step Enzyme Mix contains a specially formulated enzyme blend for both reverse transcription and PCR. Quant RTase is included in One-step Enzyme Mix and ensures highly efficient and specific reverse transcription. The RTase exhibits a higher affinity for RNA, facilitating transcription through secondary structures that inhibit other reverse transcriptases. Hot Start DNA Polymerase included in One-step Enzyme Mix provides hot-start PCR for highly specific amplification. During reverse transcription, chemically modified Hot Start DNA Polymerase is completely inactive and does Super One-step RT-PCR Kit for Virus Detection Handbook 1

3 not interfere with the reverse-transcriptase reaction. After reverse transcription, reactions are heated to 95 C for 15 min to activate Hot Start DNA Polymerase and to simultaneously inactivate the reverse transcriptases. This hot-start procedure using Hot Start DNA Polymerase eliminates extension from nonspecifically annealed primers and primer dimers in the first cycle ensuring highly specific and reproducible PCR. The 5 One-step RT-PCR Buffer is designed to enable both efficient reverse transcription and specific amplification. The buffer contains reaction buffer, dntp and One-step Enhancer Solution, which ensures high PCR efficiency in One-step RT-PCR. Important Notes 1. For high-gc and low abundance gene, adjust the reverse transcription temperature leads to high amplification efficiency. 2. An RNase-free environment should be maintained during RNA isolation and reaction setup. Consumable items related with RT-PCR should be incubated in 0.1% DEPC solution at 37 C for 12 hours and sterilized for 30min before use. 3. Super One-step RT-PCR Kit for Virus Detection is designed to be used with gene-specific primers. The use of random oligomers or oligo-dt primers is not recommended since it results in the amplification of nonspecific products. 4. Make sure the thermal cycler is preheated to 50 C before placing samples in it. 5. The primers affect RT-PCR result. GC abundance, site and length of primers should be considered. Professional software is recommended for primer design. Super One-step RT-PCR Kit for Virus Detection Handbook 2

4 Protocol 1. Thaw template RNA, primer solutions, 5 One-step RT-PCR Buffer, RNase-free ddh 2 O, centrifuge briefly and place them on ice. 2. Prepare a reaction solution according to the following table (All the steps should be performed on ice) Contents 50 µl volume 5 One-step RT-PCR Buffer 10 µl One-step Enzyme Mix 4 µl Forward Primer (10 µm) 2 µl Reverse Primer (10 µm) 2 µl RNA template 10 ng-1 µg total RNA RNase-free ddh 2 O Up to 50 µl Notes: If setting up more than one RT-PCR reaction, mix all components one time and divide into each tube. 3. Start the RT-PCR program while PCR tubes are still on ice. Wait until the thermal cycler has reached 50 C. Then place the PCR tubes in the thermal cycler. 4. Set up thermal cycler conditions according below table. Temperature Time Steps 1 50 C 30 min Reverse transcription 2 95 C 15 min Initial denaturation 3 94 C 0.5 min Denaturation C min Annealing 5 72 C min Extension Super One-step RT-PCR Kit for Virus Detection Handbook 3

5 cycles from step 3 to step 5 5. Analyze the PCR products using agarose gel electrophoresis. Ordering Information DNA Marker Product Size Cat.no. DNA Marker I 50 lanes MD MD DNA Marker III 50 lanes MD MD bp DNA Ladder 50 lanes MD MD Detection & Assay Kits Product Size Cat.no. TIANamp RNA Kit for Virus Detection 50 preps SD101 Super One-step RT-PCR Kit for Virus Detection Handbook 4

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