DNA / PCR

Volcano3G RT-PCR 2x Master Mix

 

contains all components necessary, including dNTPs and an optimized reaction buffer, for a successful and reliable real-time qPCR and RT-qPCR in all standard PCR cyclers. Included enzymes consists of a blend of our best performing, hot-start formulated, engineered Taq DNA polymerase and Volcano3G DNA polymerase. The blend has been designed to be best suited for multiplex PCRs, pathogen detection qPCRs and all kinds of probe-based qPCRs of DNA and RNA targets. 

The included Volcano3G DNA polymerase is an engineered, extremely thermostable reverse transcriptase and combined DNA polymerase, obtained through directed, artificial evolution. An aptamer-based hot-start formulation of the DNA polymerases prevent false amplification. Temperatures above 50°-55°C cause the aptamer’s secondary structure to melt and will set-free the polymerases. 

 

 

Article Product Size Price
#6101S Volcano3G RT-PCR Probe 2x Master Mix

100 reactions á 25 µl

1 x 1.25 ml

€ 120,-
#6101M Volcano3G RT-PCR Probe 2x Master Mix

500 reactions á 25 µl

5 x 1.25 ml

€ 500,-
#6201S Volcano3G RT-PCR Probe 2x Master Mix (+ROX)

100 reactions á 25 µl

1 x 1.25 ml

please inquire
#6301S Volcano3G RT-PCR Probe 2x Master Mix (+GreenDye)

100 reactions á 25 µl

1 x 1.25 ml

please inquire

 #6401S Volcano3G RT-PCR Probe 2x Master Mix (+GreenDye, +ROX)

100 reactions á 25 µl

1 x 1.25 ml

please inquire

Bulk offer: Should you require larger amounts (> 10 ml), significant discounts are available. Please contact us to receive an individual quotation.

 

download-pdf Product Manual (PDF)Order

 

 

Applications

Applications

  • Rapid detection and identification of RNA & DNA targets
  • Reverse transcription qPCRs (RT-qPCRs)
  • qPCRs (DNA or RNA targets)
  • Multiplex PCR
  • Realtime PCR
  • Probe-based PCR
  • Pathogene detection
  • Primer extensions 
  • Endpoint PCRs
  • Screening / High-throughput PCRs
  • SELEX

Example Results

coming soon

PCR protocol

Typical RT-PCR protocol

Step Temperature Time
Initial denaturation step 80°C 60 sec
Reverse Transcription step* 50°C 600 sec
PCR cycling:    
Denaturation 95°C 10 sec
Annealing/Extension** various* 50 sec   (35-50 cycles)

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).

In general, Volcano3G DNA polymerase is fully thermostable and most active between 55-95°C.

Reaction Setup

Reaction setup

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.

 

Recommended reaction setup 

Component Volume Final concentration
Primer forward (10 µM) 1.0 µl 500 nM (50-1000 nM)
Primer reverse (10 µM) 1.0 µl 500 nM (50-1000 nM)
Probe (10 µM)  x µl 50 - 1000 nM
Volcano3G RT-PCR 2x Master Mix 10 µl 1x
Template/Sample extract*  y µl  
 Nuclease-free water  up to 20 µl total reaction volume  

*= Recommended template concentration is between 0.004 ng/µl – 0.1 µg/µl (of total RNA or genomic DNA).

 

Spin down and mix all solutions carefully before use.

 

Quality Control

RT-PCR activity: Volcano3G RT-PCR Mix is tested for a successful RT-PCR performance. A 151 bp fragment (HPRT1 mRNA) is amplified from human total RNA extract in a real-time PCR cycler and visualized as a single amplified product.
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.

 

Storage

This product is shipped on cool packs. Please store the product upon arrival at -20°C.
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.

Volcano_teaser2

 

img-faqsFAQ`s

Does Volcano3G have a RNAse-H like activity?

No. Volcano3G is derived from Taq DNA polymerase, so it does not have any RNase-H activity. Reverse transcription takes place simultaneously with DNA amplification during the cycled PCR elongation step.

However, Volcano3G has a nuclease domain as the Taq wild-type enzyme and can be used for hydrolysis-based real-time PCRs. 

 

How can I limit the amplification only to mRNA targets?

Design the primers in a way that they are binding to exon-exon junctions. You can use one of the free primer design tools in the internet, such as primer-blast on the homepage: www.ncbi.nlm.nih.gov/tools/primer-blast/. Ensure, that you select the option: "primers must span an exon-exon junction". This is useful for limiting the amplification only to mRNA, as Volcano3G DNA polymerase will amplify from any nucleic acid target consisting of both RNA or DNA. However, you can use standard primers if you want to amplify mRNA as well as from the corresponding genomic DNA.

I am only getting primer dimers. How can I establish my RT-PCR?

- Keep all reagents during PCR setup on ice.

- Programm the PCR protocol on your cycler first, before you setup the PCR mix and if applicable preheat the lid of your cycler.

- When you do a new PCR for the first time, start with our standart protocol and recommended reaction setup concentrations.

- Run a temperature gradient to identify the best temperature yielding in the cleanest product. This temperature is only limited by the primer annealing temperature, as Volcano polymerase is thermostable.
- Use a "multiple primer analyzer" to test your primer sequences for self-complementarity, secondary structures and primer cross annealing.

Can I reverse transcribe long RNA-strands (>500 bp) with Volcano?

We do not recommend using Volcano3G for reverse transcription of RNA which is longer than 500 bp. Volcano3G RT-PCR Mixes are optimized for detection- and qPCR-assays with short amplicons between 60-300 bp.

I see a low-molecular band on my agarose gel - is this a contamination of the Volcano3G RT PCR Mix?

All our products are regularly checked for contamination of various kinds. However it may be possible that the aptamer, which is used for hotstart formulation may be detected as a low molecular band between 25-60 bp length.
Please contact us, should you still have reasonable doubts about the quality of your product. We are always keen to help.

 

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