PrimerdesignTM

oasigTM lyophilised
OneStep 2X RT-qPCR
Master Mix
Instructions for use of
Primerdesign oasig lyophilised OneStep Master Mix

oasig lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05

Published Date: 20 Nov 2017
Contents

Introduction 3

Kit contents 4

Kit storage 4

Suitable sample material 4

Licensing agreement and limitations of use 4

Primerdesign Ltd satisfaction guarantee 4

Quality control 4

Resuspension protocol 5

OneStep RT-qPCR detection protocol 6

OneStep RT-qPCR amplification protocol 7

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oasig lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 20 Nov 2017
Introduction
Primerdesign oasig lyophilised OneStep 2X RT-qPCR Master Mix is an optimised
complete system for use in OneStep 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 master mix
contains a thermo-stable TAQ Polymerase and MMLV as well as buffer, dNTPs, MgCl2
and stabilisers at concentrations optimised for the enzymes. Once resuspended, only
the template RNA and primer and probe mix are required to complete the experimental
set up for a perfect single tube reaction. The master mix has been freeze-dried to
produce a room temperature stable preparation.

The kit includes the lyophilised master mix, resuspension buffer and a tube of ROX dye
which can be added as required when the master mix is to be used on hardware
platforms that use ROX as a passive reference dye.

The performance of oasig lyophilised OneStep 2X RT-qPCR Master Mix is as good as

or better than leading brands. For details see www.primerdesign.co.uk

The perfect partner for genesig kits

Primerdesign oasig lyophilised OneStep 2X RT-qPCR Master Mix is designed for use
with our range of genesig qPCR pathogen detection kits. Together they represent the
ultimate solution for convenient logistics as well as high quality qPCR performance.

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oasig lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 20 Nov 2017
Kit contents
 3 x lyophilised OneStep Master Mix (50 reactions per glass ampule)

 1 x lyophilised ROX (BROWN)

 4 x Resuspension buffer (BLUE)

Kit storage
The Primerdesign oasig lyophilised OneStep 2X RT-qPCR Master Mix 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 derived RNA,
Biopsy derived RNA etc). Please ensure the samples are suitable in terms of purity,
concentration and RNA integrity. Always run at least one negative control with the
samples. To prepare a negative-control, replace the test sample with RNase/DNase
free water.

Licensing agreement and limitations of use

PCR is covered by several patents owned by Hoffman-Roche Inc and Hoffman-
LaRoche, Ltd. Purchase of Primerdesign kits does not include or provide licence with
respect to any patents owned by Hoffman-La Roche or others.

Primerdesign Ltd satisfaction guarantee

Primerdesign takes pride in the quality of all our products. Should this product fail to
perform satisfactorily when used according to the protocols in this manual,
Primerdesign will replace the item free of charge.

Quality control
As part of our ISO9001 and ISO13485 quality assurance systems, all Primerdesign
products are monitored to ensure the highest levels of performance and reliability.

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oasig lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 20 Nov 2017
Resuspension protocol
1. For each glass ampule resuspend lyophilised OneStep Master Mix in 525μl of
resuspension buffer
Do not replace the resuspension buffer with water or any other buffer.
The master mix is then ready to use as a 2X RT-qPCR master mix.

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.

 Resuspend the lyophilised ROX (BROWN) in the correct volume of

resuspension buffer (BLUE) according to table 1 below.
 Add resuspended ROX to each ampule at the correct level.

Table 1. ROX addition

ROX
ROX addition
Real-time PCR platform resuspension
per ampule
volume
Applied Biosystems 7700, 7000, and 7900,
7300 StepOne, StepOnePLUS and ViiA7 100μl 20μl
platforms, Roche capillary Lightcyclers.

All Stratagene platforms 200μl 15μl

Applied Biosystems 7500 platform 700μl 10μl

All Other machines NOT REQUIRED NOT REQUIRED

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oasig lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 20 Nov 2017
OneStep RT-qPCR detection protocol

 When using Primerdesign genesig pathogen detection kits.

For each 20µl RT-qPCR reaction add the following to each reaction tube

Components 1 Reaction
oasig lyophilised OneStep 2X RT-qPCR Master Mix 10 µl
Primer/Probe mix 1 µl
Template RNA x µl
RNase/DNase free water x µl
Final volume 20 µl

 Suggested use with user supplied primers and probe.

For each 20µl RT-qPCR reaction add the following to each reaction tube

Components 1 Reaction
oasig lyophilised OneStep 2X RT-qPCR Master Mix 10 µl
Forward primer (3pmols*) x µl
Reverse primer (3pmols*) x µl
Probe (3pmols) x µl
Template RNA x µl
RNase/DNase free water (up to Final volume) x µl
Final volume 20 µl
*3pmols of primer gives a working concentration of 150nM in a 20µl reaction

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oasig lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 20 Nov 2017
OneStep RT-qPCR amplification protocol

 For use with genesig pathogen detection kits

Step Time Temp

Reverse Transcription 10 min 55ºC
Enzyme Activation 2 min 95ºC
Denaturation 10s 95ºC
Cycling x50
DATA COLLECTION* 60s 60ºC
*Fluorogenic data should be collected during this step through the FAM and VIC channels.

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oasig lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 20 Nov 2017