HoTaq One-Step Real-Time RT-PCR Kit protocol V3.1
Contents
1. lt gt MCLAB Molecular Cloning Laboratories User Manual Version 3 1 Revision Date 09 14 2013 Product name HoTag One step Real time RT PCR Kit Cat HTRT 100 HTRT 200 Description RT PCR is widely used for measuring gene expression in tissue samples or cell culture systems Traditionally it is performed in separated two reaction steps First strand cDNA is reverse transcribed from total RNA or poly A RNA using a reverse transcriptase After then the cDNA is amplified by PCR using a DNA polymerase in another reaction MCLAB the HoTaq One step Real time RT PCR Kit offers a unique system for performing probe based real time RT PCR in a single step within a single tube with optimized reaction condition which utilizes our own proprietary engineered highly purified QuantumScript HD reverse transcriptase and hot start Taq DNA Poly merase No additional reagents or steps are required once the reaction is initiated This novel kit allows you to quantitatively detect specific RNA targets with high sensitivity unparalleled convenience and wide dynamic range The technique reduces the risk of cross contamination and minimizes reagents handling steps This method is particularly useful for applications in which the expression of a small number of genes must be analyzed in many different total RNA samples and robust amplifies high abundance transcripts from crude total RNA preparations List of Components Sufficient reagents2. are supplied in the MCLAB HoTaq One step Real time RT PCR Kit for 40 or 200 rxns Upon receipt of the kit immediately store the components at 20 C in a freezer without a defrost cycle It is recommended to prevent light exposure as little as possible 40 rxns 2x RT PCR Buffer 1 5ml x 1 25x RT PCR Enzyme Mix 40 ul x1 200 rxns 2x RT PCR Buffer 1 5ml x 1 25x RT PCR Enzyme Mix 200 ul x1 Contains regular level of ROX dNTPs and optimized buffer components for Real time PCR machines ABI 7000 7300 7700 7900 and Stratagene Mx 3000P Mx 3005P Additional Materials Required The following reagents instruments and consumables are supplied by the user e Template RNA 1 650 872 0245 www mclab com 1 e Gene specific primers and probe e DEPC treated water e Microcentrifuge e Real time thermal cycler e PCR tubes plates One step Real time RT PCR Procedure 1 Set up one step real time RT PCR reaction in a PCR tube on ice as below Component Volume Note RT PCR Buffer 12 5 uL RT PCR Enzyme Mix 1 uL Forward Primer X Final 50 to 900 nM Reverse Primer X Final 50 to 900 nM Probe X Final 50 to 250 nM RNA Template x 10 pg to 1ug total RNA Nuclease free water To 25 uL The concentration might need optimization for your specific targets For multiple reactions master mix should be made with 5 extra reagents to reduce pipette error 2 Gently mix thoroughly and then centrifuge brief
3. ly 3 Place the tube or plate in the thermal cycler and set up program using associated software Basic cycling conditions are as following One cycle at 50 C for 10 to 30 minutes One cycle at 95 C for 10 minutes Followed by 40 cycles of 95 C for 15 seconds 60 C for 1 minute 4 C hold optional Reaction time could be adjusted according RNA input Cycle number and annealing temperature are experiment dependent Troubleshooting Unexpected high or none Cq quantification cycle value e Your RNA may be degraded RNA integrity should be evaluated prior to the reaction e Your RNA sample might be contaminated with interferers for RT PCR reaction RNA purity should be evalu ated prior to the reaction Sample from crude total RNA preparation should be serial diluted as input and run multiple reactions Re precipitate or re isolate RNA from the source if necessary e Poor primers and probe design Be sure to follow guidelines when designing primers and probe for your target gene e Target gene may be at very weak expression level in your sample Paralleled control gene amplification will be helpful for troubleshooting Reference 1 Higuchi R et al 1992 BioTechnology 10 413 2 Higuchi R et al 1993 BioTechnology 11 1026 2 lt MCLAB
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