Volcano3G RT-PCR 2× Master Mix

Product#: 6101
$192.00

Size:

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

Volcano3G RT-PCR 2× Master Mix

Cat. No. 6101
Size 100 tests / 500 tests
Store -20°C

Contents

Volcano3G 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
Experimental recommendations for first use
  • Run a PCR with a temperature gradient at the annealing / extension step in order to find the optimal temperature for your assay.
  • Most RT-PCR assays work well with a short 80°C (1 min) denaturation step and a reverse transcription step at 50-55°C (10 min) followed by standard PCR cycling conditions.
Recommendations for RT-PCR/ ReactionSetup
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 25 µl total reaction volume

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

Initial denaturation step

80°C

60 sec

Reverse Transcription step*

50°C

600 sec

Annealing/Extension**

various

50 sec

Denaturation

95°C

10 sec (35-50 cycles)

Hold

<10°C

hold

 

 

A two-step as well as three-step PCR protocols can be used.
* 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). 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. DNA polymerase activity: Volcano3G DNA 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.
Licences/Patents/Disclaimers
This product is covered by the patent WO2014023318. 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 GmbH. 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. Product source: recombinant protein expression in E.coli.
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.
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.
References
  1. Volcano3G 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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