PCRMaxTM
Lyophilised OneStep
2X RT-qPCR Master Mix
Instructions for use of Lyophilised OneStep Master Mix
Lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 17 Nov 2017
Contents
Introduction 3
Kit contents 4
Kit storage 4
Suitable sample material 4
Licensing agreement and limitations of use 4
Resuspension protocol 5
OneStep RT-qPCR detection protocol 6
OneStep RT-qPCR amplification protocol 7
Lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 17 Nov 2017
2
Introduction
Our 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.
Lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 17 Nov 2017
3
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 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 HoffmanLaRoche, Ltd. Purchase of this kit does not include or provide licence with respect to
any patents owned by Hoffman-La Roche or others.
Lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 17 Nov 2017
4
Resuspension protocol
volume
Applied Biosystems 7700, 7000, and 7900,
7300 StepOne, StepOnePLUS and ViiA7
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
Real-time PCR platform
ROX
resuspension
ROX addition
per ampule
platforms, Roche capillary Lightcyclers.
All Stratagene platforms
Applied Biosystems 7500 platform
All Other machines
100μl 20μl
200μl 15μl
700μl 10μl
NOT REQUIRED NOT REQUIRED
Lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
5
Published Date: 17 Nov 2017
OneStep RT-qPCR detection protocol
Components
1 Reaction
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
Components
1 Reaction
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
When using PCRmax qPCR test kits.
For each 20µl RT-qPCR reaction add the following to each reaction tube
Suggested use with user supplied primers and probe.
For each 20µl RT-qPCR reaction add the following to each reaction tube
*3pmols of primer gives a working concentration of 150nM in a 20µl reaction
Lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 17 Nov 2017
6
OneStep RT-qPCR amplification protocol
Step
Time
Temp
Reverse Transcription
10 min
55ºC
Enzyme Activation
2 min
95ºC
Denaturation
10 s
95ºC
DATA COLLECTION*
60 s
60ºC
For use with PCRmax qPCR test kits
Cycling x50
*Fluorogenic data should be collected during this step through the FAM channel.
7
Lyophilised OneStep 2X RT-qPCR Master Mix Handbook HB04.04.05
Published Date: 17 Nov 2017
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