RNA / RT-PCR

Volcano2G RT-PCR 2x Master Mix

 

is available in four different ready-to-use formats. It can be used for all real-time dyes as well as probe-based qPCR assays, optionally containing ROX as a passive reference dye. Volcano2G RT-PCR 2x Master Mixes contain all components necessary for a successful and reliable qPCR in all standard real-time PCR cyclers. Only primers and template need to be added. These mixes provide robust PCR performance for a wide range of qPCR applications. Volcano2G is the next generation of the original Volcano DNA polymerase with further improved reverse transcription activity. This enzyme is an engineered, extremely thermostable reverse transcriptase and combined DNA polymerase, obtained through directed, artificial evolution.
Volcano2G DNA polymerase facilitates "zero-step" RT-PCRs directly from RNA templates (without an isothermal reverse transcription step), as reverse transcription takes place simultaneously with DNA amplification during the cycled PCR elongation step. This also allows reverse transcription reactions at high temperatures, thus minimizing the problems encountered with strong secondary structures in RNA that melt at elevated temperatures.

 

 

ArticleProductSizePrice
#6100SVolcano2G RT-PCR Probe 2x Master Mix100 reactions ├í 25 ┬Ál, 1 x 1.25 mlÔéČ 150,-
#6100MVolcano2G RT-PCR Probe 2x Master Mix500 reactions ├í 25 ┬Ál, 5 x 1.25 mlÔéČ 650,-
#6200SVolcano2G RT-PCR Probe 2x Master Mix (+ROX)100 reactions ├í 25 ┬Ál, 1 x 1.25 mlÔéČ 150,-
#6200MVolcano2G RT-PCR Probe 2x Master Mix (+ROX)500 reactions ├í 25 ┬Ál, 5 x 1.25 mlÔéČ 650,-
#6300SVolcano2G RT-PCR GreenDye 2x Master Mix100 reactions ├í 25 ┬Ál, 1┬áx 1.25 mlÔéČ 150,-
#6300MVolcano2G RT-PCR GreenDye 2x Master Mix500 reactions ├í 25 ┬Ál, 5┬áx 1.25 mlÔéČ 650,-
#6400SVolcano2G RT-PCR GreenDye 2x Master Mix (+ROX)100 reactions ├í 25 ┬Ál, 1 x 1.25 mlÔéČ 150,-
#6400MVolcano2G RT-PCR GreenDye 2x Master Mix (+ROX)500 reactions ├í 25 ┬Ál, 5 x 1.25 mlÔéČ 650,-

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 targets
  • Reverse transcription PCRs (RT-PCRs)
  • Real-time RT-PCRs
  • SELEX
  • Primer extensions

Example Results

Volcano qPCR Mixes are available for real-time dye as well as probe-based qPCR assays:

Probe-based qPCRs:

NONO mRNA was detected using a 10-fold dilution series of total RNA by using a probe-based qPCR assay and the zero-step RT-PCR protocol:

 

ACTB mRNA was detected using a 10-fold dilution series of total RNA by using a probe-based qPCR assay and the zero-step RT-PCR protocol:

 

GAPDH mRNA was detected using a 10-fold dilution series of total RNA by using a probe-based qPCR assay and the zero-step RT-PCR protocol:

 

GreenDye-based qPCRs:

HPRT1 mRNA was detected using a 10-fold dilution series of total RNA by using a GreenDye-based real-time qPCR assay and the zero-step RT-PCR protocol:

 

Easier and faster: 0-step RT-PCR protocol

RT-PCR-protocols2

Skip the reverse transcription step. No isothermal reverse-transcription steps are needed. 

Volcano2G is a PCR-active reverse transcriptase and extremely thermostable with a half-life at 95°C of >40min! 

Reaction Setup

Reaction setup

ComponentVolumeFinal concentration
Volcano2G RT-PCR 2x Master Mix10┬á┬Ál1x
Primer forward (10 ┬ÁM)1 ┬Ál500 nM┬á(50-1000┬ánM)
Primer reverse (10 ┬ÁM)1 ┬Ál500 nM (50-1000 nM)
Template/Sample extract*1-8 ┬Ál┬á>1 ng (1-1000 ng)
Nuclease-free waterup to 20┬Ál total reaction volume

Keep all components on ice.
Spin down and mix all solutions carefully before use.

Please note that Volcano RT-PCR 2x Master Mix is amplifying from DNA and RNA templates. So please use DNA-free RNA samples or use RNA-selective primers, which are binding onto exon-exon junctions.

* Suggested final template concentration should be from┬á1 ng/┬Ál - 50 ng/┬Ál (total RNA).┬á

Zero-step RT-PCR

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

StepTemperatureTime
Initial denaturation95┬░C2 min
Denaturation95┬░C15 sec
Annealing/Extension*various45 sec   (25-40 cycles)
Hold<10┬░Chold

A two-step as well as three-step PCR protocols can be used.
* A new 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.

 

Detailed product description

The pre-ready 2x mix ensures repeatable RT-qPCR results, significantly reduces set-up times and the risk of pipetting mistakes. Only primers and template need to be added. The reaction buffer of the Volcano RT-PCR 2x Master Mix is optimized for amplicon size between 60-300 bp.

Volcano RT-PCR 2x Master Mix is available for probe-based assays as well as for real-time dye applications.

 

Quality Control

RT-PCR activity: Volcano 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: Volcano 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 Volcano have a RNAse-H like activity?

No. Volcano 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, Volcano 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 Volcano 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 Volcano for reverse transcription of RNA which is longer than 500 bp. Volcano and the ready-to-use Volcano Mixes are optimized for detection- and qPCR-assays with short amplicons between 60-200 bp.

 

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