Volcano2G RT-PCR Probe 2x Master Mix

Product#: 6100
$240.00

Size:

  • 100 Tests
  • 500 Tests
Availability:
Ships in 1-2 weeks

Volcano2G RT-PCR Probe 2x Master Mix

Cat. No. 6100
Size 100 tests / 500 tests
Store -20 oC

Contents

Volcano2G RT-PCR 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. Hot-start formulation of Volcano DNA polymerase prevents false amplification during the reaction set-up.
#6100 Volcano2G RT-PCR Probe 2x Master Mix is suitable for all probe-based qPCR assays.
#6200 Volcano2G RT-PCR Probe 2x Master Mix (+ROX) is suitable for all probe-based qPCR assays requiring a passive reference dye.

Applications
  • Rapid detection and identification of RNA targets
  • Reverse transcription PCRs (RT-PCRs)
  • Real-time RT-qPCRs
  • qPCRs
Safety
This product does not require a Material Safety Data Sheet because it does neither contain more than 1% of a component classified as dangerous or hazardous nor more than 0.1% of a component classified as carcinogenic. However, we generally recommend the use of gloves, lab coats and eye protection when working with these or any other chemical reagents. myPOLS Biotec takes no liability for damage resulting from handling or contact with this product. Further information can be found in the REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL

Important notes
  • Volcano2G RT-PCR 2x Master Mix is optimized for an amplicon    size    between    60- 300    bp.
  • Volcano2G RT-PCR 2x  Master Mix is available for all real-time probe-based assays as well as GreenDye-based assays, both with and without     ROX as a passive reference dye.
  • 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.
Licences/Patents/Disclaimers
This product is covered by a pending patent application. It is for the purchaser’s own internal research use and may not be resold, modified or used for production and commercial purposes of any kind without an agreement with myPOLS Biotec. For information on obtaining additional rights, please contact: info@mypols.de. The product is for research use only and may be used for invitro experiments only.
 
Quality Control Assays RT-PCR activity
Volcano2G RT-PCR Mix is tested for a successful RT-qPCR performance. A 151 bp fragment (HPRT1 mRNA) is amplified from human total RNA extract and linearity between different template dilutions confirmed. DNA polymerase activity: Volcano2G polymerase activity 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 info@mypols.de for the lot-specific concentration. No contamination has been detected in standard test reactions.

Recommendations for PCR/ Reaction Setup
 
PCR Mix

Component

Volume

Final concentration

Volcano2G RT-PCR 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)

Template/Sample extract

* x µl

>1 ng (1-1000 ng)

Nuclease-free water

up to 25µl total reaction vol.

Keep all components on ice. Spin down and mix all solutions carefully before use.
* Suggested template concentration should be 0.1 ng/µl - 1 µg/µl (total RNA).

Typical 0-step RT-PCR protocol
(an isothermal reverse transcription step is not needed)

Initial denaturation

95°C

2 min

Denaturation

95°C

15 sec

Annealing/Extension*

Various

45 sec (25-40 cycles)

Hold

<10°C

hold

A two-step as well as three-step PCR protocols can be used.
*A new RT-PCR is ideally established by running a temperature gradient in order to find the best 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). Volcano2G DNA polymerase is most active between 50-95°C.

References
  1. Volcano2G DNA polymerase is based on: Structure and Function of an RNA-Reading Thermostable DNA Polymerase. Angew. Chem. Int. Ed.,2013; 52:11935–11939.Blatter, N.,Bergen, K.,Nolte, O.,Welte, W.,Diederichs, K.,Mayer, J.,Wieland,M.and Marx, A.

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