PB25.41 qPCRBIO Probe 1 Step Go Lo ROX v1.8

PB25.
41
www.pcrbio.com
qPCRBIO Probe
1-Step Go Lo-ROX
Product description: 2x qPCRBIO Probe

20x RTase Go
Pack size (with RNase
1-Step Go Lo-ROX
qPCRBIO Probe 1-Step Go is a universal inhibitor)
probe kit designed for fast, highly specific 100 reactions 1 x 1 mL 1 x 100 µL
and ultra-sensitive RT-qPCR. The latest 300 reactions 3 x 1 mL 3 x 100 µL
developments in reverse transcriptase
500 reactions 1 x 5 mL 1 x 500 µL
technology and buffer chemistry are used
1200 reactions 12 x 1 mL 12 x 100 µL
to give efficient cDNA synthesis and real-
time PCR in a single tube. 5000 reactions 1 x 50 mL 1 x 5 mL
50000 reactions 1 x 500 mL 1 x 50 mL

The kit is engineered for use on a wide range of
probe technologies such as TaqMan®, Scorpions®
and molecular beacon probes. It can be used to
quantify any RNA template including mRNA,
Shipping and storage
total RNA and viral RNA sequences. qPCRBIO On arrival the kit should be stored between -30 °C
Probe 1-Step Go is designed to give rapid and and -15 °C. Avoid prolonged exposure to light. If
accurate results over a broad range of template stored correctly the kit will retain full activity for
concentrations and is ideally suited to the 12 months. Avoid exposure of the stock solution to
detection of RNA viruses including SARS-CoV-2. frequent temperature changes and limit handling
at room temperature to the necessary minimum.
The kit includes a thermostable and extremely
Do not store the mix once it is combined with the
active modified MMLV reverse transcriptase (RTase
RTase.
Go) and advanced RNase inhibitor that prevents
degradation of RNA by contaminating RNase.
Antibody-mediated hot start technology prevents Limitations of product use
the formation of primer dimers and non-specific The product may be used for in vitro research
products giving highly specific and ultra-sensitive purposes only.
real-time RT-PCR with unrivalled efficiency in
multiplex.
Technical support
High-throughput screening has resulted in a
Help is available on our website at https://pcrbio.
buffer system that allows efficient amplification
com/resources/ including answers to frequently
from GC-rich and AT-rich templates, under fast
asked technical questions. For technical support
and standard cycling conditions.
and troubleshooting please email technical@
pcrbio.com with the following information:
• Amplicon size
• Reaction setup
• Cycling conditions
• Screen grabs of amplification traces and
melting profile
Important considerations
Instrument compatibility: Different real-time PCR instruments require different levels of ROX passive
reference. Generally, modern instruments do not require passive reference but include the option to use
it for normalisation. Please check our qPCRBIO Selection Tool to determine which ROX concentration your
instrument requires (https://pcrbio.com/resources/qpcr-selection-tool/).
Primer design: For efficient amplification under fast cycling conditions we recommend amplicon
lengths between 80 bp and 200 bp. With all manufacturers, the shorter the amplicon length,
the faster the reaction can be cycled. Amplicon lengths should not exceed 400 bp. Primers
should have a predicted melting temperature of around 60 °C, using default Primer 3 settings
(http://bioinfo.ut.ee/primer3/). For TaqMan® probes, choose a probe close to the 5’ primer and avoid
terminal guanosine residues.
Template concentration: As target copy number will vary, it is important to select the correct template
concentration to correctly quantify the target sequence. A good concentration will display clear
separation between amplification curves (Fig.1). At lower template concentrations, the amplification
curves will begin to group together and Ct values will not fit the standard curve (Fig.2).
Fig.1 Fig.2
Reaction setup
1. Before starting, briefly vortex 2x qPCRBIO Probe 1-Step Go Mix.
2. Prepare a master mix based on the following table. We also recommend setting up a no-RTase control:
Reagent 20 µL reaction Final conc. Notes
2x qPCRBIO Probe 1-Step Go Mix 10 µL 1x
Forward primer (10 µM) 0.8 µL 400 nM
See above for optimal primer design
Reverse primer (10 µM) 0.8 µL 400 nM
Probe (10 µM) 0.4 µL 200 nM
0.2 µL for sensitive SARS-CoV-2 detection.
20x RTase Go 0.2 µL 0.2x
Alternatively, titrate down to 0.05 μL.
Addition of sample as 2 to 5 μL volumes will

Viral RNA: 10 to 1x108 copies
improve assay precision. 5 μL of swab extract
Template RNA Total RNA: 1 pg to 1 µg Variable
is recommended for SARS-CoV-2 diagnostic
mRNA: >0.01 pg
assays
PCR grade dH2O Up to 20 µL final volume
3. Program the instrument using the following conditions, acquiring data on the appropriate channel:
Cycles Temperature Time Notes
Reverse transcription: 45 °C is recommended for most applications.

1 45 °C to 55 °C 10-20 minutes 55 °C should be used only when amplicon contains regions of high
secondary structure
1 95 °C 2 minutes Polymerase activation
Denaturation
95 °C 5 seconds
40 Anneal/Extension: do not exceed 30 seconds, do not use temperatures
60 °C to 65 °C 20-30 seconds
below 60 °C
Melt analysis Refer to instrument instructions Optional melt profile analysis, available for hybridisation probes only
version 1.8

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PB25.41 qPCRBIO Probe 1 Step Go Lo ROX v1.8

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PB25.

Product description: 2x qPCRBIO Probe

50000 reactions 1 x 500 mL 1 x 50 mL

Addition of sample as 2 to 5 μL volumes will

PCR grade dH2O Up to 20 µL final volume

Cycles Temperature Time Notes

Reverse transcription: 45 °C is recommended for most applications.

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