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.
|#8100S||Volcano2G DNA polymerase||¬†250¬†U, 5U/¬Ķl, 50 ¬Ķl||‚ā¨ 90,-|
|#8100M||Volcano2G DNA¬†polymerase||1000 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.
Easier and faster: 0-step RT-PCR protocol:
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:
- Rapid detection and identification of RNA targets
- Reverse transcription PCRs (RT-PCRs)
- Real-time RT-PCRs
- Primer extensions
|Primer forward (10 ¬ĶM)||1 ¬Ķl||500 nM¬†(50-1000¬†nM)|
|Primer reverse (10 ¬ĶM)||1 ¬Ķl||500 nM (50-1000 nM)|
|dNTPs (10 mM)||2¬†¬Ķl||400 ¬ĶM|
|5 x Volcano¬†buffer||10¬†¬Ķl||1x|
|Volcano2G DNA polymerase¬†5 U/¬Ķl||1 ¬Ķl||5-10 U / reaction|
|Template/Sample extract*||various¬†¬Ķl||¬†>1 ng (1-1000 ng)|
|Nuclease-free water||up 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.¬†
Typical 0-step RT-PCR protocol (an¬†isothermal reverse transcription step is not needed)
|Initial denaturation||95¬įC||2 min|
|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).
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 firstname.lastname@example.org for the lot-specific concentration.
This product is shipped on cool packs. Please store the product upon arrival at -20¬įC.
How can I limit the amplification only to mRNA targets?
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?
- 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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