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Volcano2G DNA polymerase

 

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 ‚Äúzero-step‚ÄĚ 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
#8100SVolcano2G DNA polymerase¬†250¬†U, 5U/¬Ķl, 50 ¬Ķl‚ā¨ 90,-
#8100MVolcano2G DNA¬†polymerase1000 U, 5U/¬Ķl, 200 ¬Ķl‚ā¨ 300,-

Bulk offer: Should you require larger amounts (> 2000 U), significant discounts are available. Please contact us to receive an individual quotation.

 

download-pdf Product Manual (PDF)Order

 

 

Example Results

Easier and faster: 0-step RT-PCR protocol:

RT-PCR-protocols2

 

Skip the reverse transcription step. Isothermal reverse-transcription steps are not needed. 

Volcano2G is a PCR-active reverse transcriptase and extremely thermostable with a¬†half-life at 95¬įC of >40 min.

Volcano2G DNA polymerase can also be used for real-time PCR, when adding a real-time PCR dye. Volcano2G has a Taq DNA polymerase-like nuclease domain making it also suitable for hydrolysis probe based assays. As demonstrated here, 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:

 

NONO-qPCR-assay

 

 

Applications

Applications

  • Rapid detection and identification of RNA targets
  • Reverse transcription PCRs (RT-PCRs)
  • Real-time RT-PCRs
  • SELEX
  • Primer extensions

Reaction Setup

Reaction setup

ComponentVolumeFinal concentration
Primer forward (10 ¬ĶM)1 ¬Ķl500 nM¬†(50-1000¬†nM)
Primer reverse (10 ¬ĶM)1 ¬Ķl500 nM (50-1000 nM)
dNTPs (10 mM)2¬†¬Ķl400 ¬ĶM
5 x Volcano¬†buffer10¬†¬Ķl1x
Volcano2G DNA polymerase¬†5 U/¬Ķl1 ¬Ķl5-10 U / reaction
Template/Sample extract*various¬†¬Ķl¬†>1 ng (1-1000 ng)
Nuclease-free waterup to 50 ¬Ķl total reaction volume

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

* Recommended¬†final template concentration is¬†between 5-50 ng/¬Ķl.¬†

Zero-step RT-PCR

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

StepTemperatureTime
Initial denaturation95¬įC2 min
Denaturation95¬įC15 sec
Annealing/Extension*various*45 sec   (25-40 cycles)
Optional melting step

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 polymerease is most active between 50-95¬įC.

 

Detailed product description

Volcano2G 5x reaction buffer is optimized for amplicon size between 60-300 bp.

Volcano2G reverse transcriptase can also be used for realtime PCR, when adding a suitable realtime PCR dye or a fluorescent probe.
This unique enzyme enables PCR-based RNA detection without a separate reverse transcription step.
Volcano2G reverse transcriptase can reliably detect RNA targets from ‚Č• 1 ng of total RNA input.

Volcano2G DNA polymerase does not require Mn2+ for optimal activity. However, some assays may be further optimized by the addition of Mn2+ to the reaction (suggested range is 0.5 - 1 mM, Mn2+ not provided).

 

Quality Control

RT-PCR activity: Volcano2G DNA polymerase activity was tested for a successful RT-PCR performance. A 151 bp fragment (HPRT1 mRNA) was amplified from human total RNA extract in a PCR cycler and visualized as a single amplified product.
DNA polymerase activity: Volcano2G polymerase activity has been monitored and adjusted to a specific DNA polymerase activity using an artificial DNA template and DNA primer.
Enzyme-concentration has been 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¬įC.

 

Volcano_teaser2

 

img-faqsFAQ`s

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.

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. 

 

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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