Volcano3G RT-PCR Probe 2x Master Mix

Volcano3G RT-PCR Probe 2x Master Mix

This Direct RT-PCR MasterMix can be used for the detection of diferent kinds of virus such as COVID- 19. Samples can be obtained from Dry Swabs or Gargle

Catalog Number: #6101

Size: 100 tests/rxns

Also Known As:  COVID-19 Direct PCR, SARS-CoV-2, Direct RT-qPCR

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Contents

This RT-PCR Probe 2x Master Mix contains all
components necessary for a successful and reliable real-time
RT-qPCR in all standard PCR cyclers, including dNTPs and an
optimized reaction buffer.
An aptamer-based hot-start formulation of the Volcano3G DNA
polymerase prevents false amplification. Temperatures above
50°C cause the aptamer’s secondary structure to melt and will
set-free the polymerase.

Applications

 Rapid detection and identification of RNA & DNA targets
 Reverse transcription qPCRs (RT-qPCRs)
 qPCRs

qpcr-instruments

Our RT-qPCR Kit Is Fully Compatible With:

    • ABI 7500
    • Bio-Rad CFX96, CFX384
    • Bio-Rad Touch / iQ5
    • Cepheid Smartcycler/Smartcycler II
    • 7 QuanStudio’s 6, 7,  12k
    • Roche: LightCycler®480/1536/Nano
    • Agilent: Mx3005p, Mx3000P
    • Qiagen: RotorGene6000,
    • QEppendorf Mastercycler
    • And More…

We Have Different Kind Of ROX Availabe:

  • Low ROX,

  • High ROX

  • NO ROX

This means that our Master Mix can be adapted to be compatible with more PCR Thermocyclers.

Experimental recommendations for first use:

– Run a PCR with a temperature gradient at the RT-step and
annealing step in order to find the optimal temperature for
your assay.
– Most RT-PCR assays work well with a RT-cycling step
consisting of a short denaturation followed by incubation at
58-70°C and subsequent PCR cycling

covid-19-test-tube
laboratory-materials

Materials and Devices Required but Not Provided:

RT-PCR/PCR Mix
Component                                                       Volume          Final concentration
Volcano3G RT-PCR Probe 2x Master Mix    12.5 µl                        1x
Primer forward (10 µM) 1.25 µl                      500 nM               (50-1000 nM)
Primer reverse (10 µM) 1.25 µl                       500 nM               (50-1000 nM)
Probe (10 µM) x µl                                    50-1000 nM
Template/Sample extract*                                   y µl                 >0.1 ng (0.1-2500 ng)
Nuclease-free water up to 25µl total reaction vol.
Keep all components on ice.
Spin down and mix all solutions carefully before use.
* Recommended template concentration should be 0.004 ng/µl – 0.1 µg/µl (of total RNA
or genomic DNA).

Typical RT-PCR protocol

RT cycling(10 cycles)
Denaturation                                    95°C            3 sec
Reverse transcription*                   58-70°C      60 sec
(temperature to be optimized)

PCR cycling                                 (35-50 cycles)
Denaturation                                    95°C             10 sec
Annealing/Extension**                  58-70°C       50 sec
Hold <10°C hold

* Volcano3G DNA polymerase allows “zero-step” RT-PCRs directly from RNA templates (without an
isothermal reverse transcription step), as reverse transcription also takes place simultaneously with
DNA amplification during the cycled PCR elongation step. Thus a reverse transcription step is optional
and can be omitted in some cases.
**A new RT-PCR is ideally established by running a temperature gradient in order to find the best
reverse transcription / annealing / extension temperature for each primer pair. The annealing
temperature of a primer is strongly influenced by its nucleic acid sequence and the reaction buffer
composition (salts and pH). Since RNA:DNA hybrids are typically more stable than DNA:DNA hybrids, the
annealing temperature in the RT-step can be higher than in during PCR cycling.
Volcano3G DNA polymerase is fully thermostable and most active between 55-95°C

Quality Control Assays

RT-PCR activity: Volcano3G RT-PCR Probe Mix is tested for a
successful RT-qPCR performance. A 151 bp fragment (HPRT1
mRNA) is amplified from human total RNA extract and the
linearity of amplification over a specified serial dilution is
demonstrated. The activity of the Volcano3G DNA polymerase
is monitored and adjusted to a specific DNA polymerase
activity using an artificial DNA template and DNA primer.
Enzyme concentration is determined by protein-specific
staining. Please inquire more information at [email protected]
for the lot-specific concentration.
No contamination has been detected in standard test reactions

Important notes
 Volcano3G RT-PCR Probe 2x Master Mix works very well
also for DNA amplification assays
 This master mix is optimized for an amplicon size between
60- 300 bp.
 Minimize the number of freeze-thaw cycles by storing in
aliquots. For a day-to-day use, we recommend keeping an
aliquot at 4°C.

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