Volcano3G RT-PCR Probe 2x Master Mix

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.

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

• • 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.

– 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

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).

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