Techne lyophilised One Step qRT-PCR MasterMix
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Techne lyophilised One Step qRT-PCR MasterMix Instructions for use of Techne Prime Pro lyophilised One Step MasterMix

Contents Introduction Suitable Sample Material Licensing Agreement and Limitations of Use Techne Satisfaction Guarantee Quality Control Bench-side Protocol Reaction setup Amplification Protocol 2 Techne Lyophilised one step MasterMix from Bibby Scientific

Introduction Techne Prime Pro qRT-PCR MasterMix is an optimised complete system for use in ONE STEP real-time PCR. Removal of a separate Reverse Transcription step, reduces handling errors, and greatly reduces the time taken to obtain results. The product is stable at ambient temperatures for at least 18 months and can be conveniently shipped and stored at room temperature. The MasterMix contains a thermo-stable TAQ Polymerase and MMLV as well as buffer, MgCl2 and stabilisers at concentrations optimised for the enzymes. Once reconstituted, only the template RNA and primer and probe mix are...

3 x Lyophilised MasterMix (50 reactions per glass ampule) 4 x Re-suspension buffer (BLUE) Kit Storage The Techne Lyophilised One Step MasterMix should be stored at ambient temperature on arrival. The kit is stable for at least 18 months at ambient temperature. Once resuspended in the provided buffer the kit should be stored at -20°C. Repeated freeze/thawing will not compromise the performance of the product. Under these conditions reagents are stable for six months from date of resuspension. Suitable Sample Material All kinds of RNA sample material can be used (e.g. Viral RNA, cell culture...

Bench-side Protocol 1. For each glass ampule; Re-suspend lyophilised One Step MasterMix in 525µl of resuspension buffer Do not replace the re-suspension buffer with water or any other buffer. The MasterMix is then ready to use as a 2X qPCR MasterMix. 2. Add ROX if required ROX is required for platforms that use ROX as a passive reference guide. Use table 1 below to see if ROX addition is required for your hardware platform. If ROX is required then follow the instructions below. • • Re-suspend the Lyophilised ROX (BROWN) in the correct volume of re-suspension buffer(BLUE) according to table...

Reaction setup • When using Techne pathogen detection kits. For each 20µl real-time PCR reaction add the following to each reaction tube Components Techne Lyophilised One Step MasterMix Primer/Probe mix Template RNA RNAse/DNAse free water Final volume Suggested use with user supplied primers and probe. For each 20µl real-time PCR reaction add the following to each reaction tube Components 1 Reaction Techne Lyophilised One Step MasterMix 10 µl Primers (3pmols Forward and Reverse) x µl Probe (3pmols) x µl Template RNA x µl RNAse/DNAse free water (up to Final volume) x µl Final volume 20 µl...

Amplification Protocol • For use with Double Dye (TaqMan® gene detection chemistry) Step Reverse Transcription Enzyme Activation - Hot Start Denaturation DATA COLLECTION* *Fluorogenic data should be collected during this step according to the detection kit. For use with SYBR® Green detection chemistry Step Reverse Transcription Enzyme activation - Hot Start Denaturation DATA COLLECTION* Melt Curve** *Fluorogenic data should be collected during this step through the SYBR® Green channel. **A post PCR run melt curve can be used to prove the specificity of the primers. See the manufactures...