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┬áthe original Volcano┬áDNA polymerase with further improved reverse transcription activity. This enzyme┬áis┬áan engineered, extremely┬áthermostable 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┬áalso allows reverse transcription reactions at high temperatures, thus minimizing the problems encountered with strong secondary structures in RNA that melt at elevated temperatures.
|#6100S||Volcano2G RT-PCR Probe 2x Master Mix||100 reactions ├á 25 ┬Ál, 1 x 1.25 ml||ÔéČ 150,-|
|#6100M||Volcano2G RT-PCR Probe 2x Master Mix||500 reactions ├á 25 ┬Ál, 5 x 1.25 ml||ÔéČ 650,-|
|#6200S||Volcano2G RT-PCR Probe 2x Master Mix (+ROX)||100 reactions ├á 25 ┬Ál, 1 x 1.25 ml||ÔéČ 150,-|
|#6200M||Volcano2G RT-PCR Probe 2x Master Mix (+ROX)||500 reactions ├á 25 ┬Ál, 5 x 1.25 ml||ÔéČ 650,-|
|#6300S||Volcano2G RT-PCR GreenDye 2x Master Mix||100 reactions ├á 25 ┬Ál, 1┬áx 1.25 ml||ÔéČ 150,-|
|#6300M||Volcano2G RT-PCR GreenDye 2x Master Mix||500 reactions ├á 25 ┬Ál, 5┬áx 1.25 ml||ÔéČ 650,-|
|#6400S||Volcano2G RT-PCR GreenDye 2x Master Mix (+ROX)||100 reactions ├á 25 ┬Ál, 1 x 1.25 ml||ÔéČ 150,-|
|#6400M||Volcano2G RT-PCR GreenDye 2x Master Mix (+ROX)||500 reactions ├á 25 ┬Ál, 5 x 1.25 ml||ÔéČ 650,-|
Bulk offer: Should you require larger amounts (> 10 ml), significant discounts are available. Please contact us to receive an individual quotation.
- Rapid detection and identification of RNA targets
- Reverse transcription PCRs (RT-PCRs)
- Real-time RT-PCRs
- Primer extensions
Volcano qPCR Mixes are available for real-time dye as well as probe-based qPCR assays:
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:
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
Skip the reverse transcription step. No isothermal reverse-transcription steps are needed.┬á
Volcano2G is a PCR-active reverse transcriptase and extremely thermostable with a┬áhalf-life at 95┬░C of >40min!┬á
|Volcano2G RT-PCR 2x Master Mix||10┬á┬Ál||1x|
|Primer forward (10 ┬ÁM)||1 ┬Ál||500 nM┬á(50-1000┬ánM)|
|Primer reverse (10 ┬ÁM)||1 ┬Ál||500 nM (50-1000 nM)|
|Template/Sample extract*||1-8 ┬Ál||┬á>1 ng (1-1000 ng)|
|Nuclease-free water||up 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
Typical 0-step RT-PCR protocol (an┬áisothermal reverse transcription step is not needed)
|Initial denaturation||95┬░C||2 min|
|Annealing/Extension*||various||45┬ásec ┬á (25-40 cycles)|
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┬áVolcano RT-PCR 2x Master Mix is optimized for┬áamplicon size between 60-300 bp.
Volcano RT-PCR 2x Master Mix is available┬áfor probe-based assays as well as for real-time dye applications.
RT-PCR activity: Volcano RT-PCR Mix┬áis tested for a successful RT-PCR performance. A 151┬ábp 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┬ápolymerase 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 firstname.lastname@example.org for the lot-specific concentration.
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.
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.┬áReverse 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.┬á┬á
How can I limit the amplification only to mRNA targets?
I am only getting primer dimers. How can I establish my RT-PCR?
- 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┬álimited by the primer annealing┬átemperature, as Volcano polymerase is thermostable.
- Use a "multiple primer analyzer"┬áto 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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