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Volcano3G® RT-PCR Probe 2x Master Mix
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Volcano3G® RT-PCR Probe 2x Master Mix contains all components required for general RT-qPCR
- optimized reaction mix for sensitive and reliable results
- engineered, truly thermostable Taq DNA polymerases with reverse transcriptase activity
- fast start function due to a hotstart aptamer formulation which prevents unspecific amplification at lower temperatures (<57°C)
- Pipetting aid in form of a blue dye for better visibility during aliquoting into well-plates
- included lysis function** = A sample extraction and sample lysis is not necessary as immediate hot PCR cycling conditions can brake cell- and virus membranes. Please see our published references to this product.
Note: Volcano3G® RT-PCR Probe 2x Master Mix is a unique master mix. If you are using it for the first time, we recommend to design primers to have higher melting/annealing temperatures (above 65°C). Additionally, for assay setup it is advised to perform RT-PCR test reactions with temperature gradients (i.e., for the annealing and extension steps) to identify the most proficient PCR protocol for your needs using our Volcano DNA polymerase products.
For research use and further manufacturing.
In case you are aiming to use our RUO products as components or for your development of e.g. an IVD medical device, please contact us.
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- Rapid detection and identification of RNA & DNA targets
- Reverse transcription qPCRs (RT-qPCRs)*
- Direct from cell RT-PCRs*
- qPCRs
*Please see for example publication regarding
Corona virus (SARS-CoV-2, COVID-19) detections from crude oral swabs: (Detection of SARS-CoV-2 from raw patient samples by coupled high temperature reverse transcription and amplification)
or qPCRs from Cell cultures: (Open access: Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription: a ‘zero-step’ RT-qPCR protocol)Variants of Volcano3G® RT-PCR 2x Master Mix are also available with a DNA binding dye (GreenDye), with or without passive reference dye (ROX)(#6301/#6401)
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Please contact us if you have questions for this product.
As Volcano3G is a very unique mastermix, it is not trivial to get started. We have already established PCRs for other customers and offer this service for free to you too.
How do I have to store the mix?
- Keep at -20°C and minimize the number of freeze-thaw cycles by storing the product in aliquots. For a day-to-day use, we recommend keeping an aliquot at 4°C.
Can I use the mix with crude samples for direct PCRs?
Yes this is possible in general. We have for example tested Volcano with cells and cell-lysates as well as unpurified oral swab samples. More information can be found in the download section of this product.
How stable is the mix?
Volcano3G® RT-PCR Probe 2x Master Mix is shipped on cool-packs around the world. It has been stress-tested at long time periods at elevated temperatures (up to 40°C) without any loss of detectable activity. However we recommend to follow our storage recommendations-
Can I simply replace my currently used RT-PCR mix with your Volcano3G® RT-PCR Probe 2x Master Mix?
No. We strongly recommend to design primers to have higher melting/annealing temperatures (above 65°C). Additionally, for assay setup it is advised to perform RT-PCR test reactions with temperature gradients (i.e., for the annealing and extension steps) to identify the most proficient PCR protocol for your needs using our Volcano DNA polymerase products.
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- Download Volcano3G RT-PCR Probe 2x Master Mix Manual
Publication using Volcano3G® for detection of the Corona virus without sample extraction:
Detection of SARS-CoV-2 from raw patient samples by coupled high temperature reverse transcription and amplification.
Kuiper JWP, Baade T, Kremer M, Kranaster R, Irmisch L, Schuchmann M, Zander J, Marx A, Hauck CR.
PLoS One. 2020 Nov 2;15(11):e0241740
Link: https://doi.org/10.1371/journal.pone.0241740 -
Volcano3G® RT-PCR Probe 2x Master Mix is tested for a successful, reproducible RT-qPCR performance. Enzyme concentrations are determined by protein-specific staining.
The activities of the enzymes are monitored and adjusted to a specific DNA polymerase activity using a synthetic DNA template and DNA primer.
Additionally, a 151 bp fragment (HPRT1 mRNA) is amplified from an in-vitro transcribed RNA strand by a 0-step real-time RT-qPCR protocol using hydrolysis probes. The linearity of amplification over a specified serial dilution is demonstrated.
No human RNA and DNA as well as no nuclease contaminations is detected.
This product is for research use and further manufacturing. In case you are aiming to use our RUO products as components or for your development of e.g. an IVD medical device, please contact us.
Establishing RT-PCR assays with Volcano3G® RT-PCR Probe 2x Master Mix:
1. Make sure that your primers have melting points higher than 65°C
2. Optimize PCR-steps: Prepare one big PCR reaction with cDNA and aliquot it into the plate in multiple wells. Apply a two-step PCR protocol (for example 95°C 10 sec. and a temperature gradient between 57-67°C (1min, 40 cycles).
> From this experiment you can select the best temperature/well which shows the earliest Ct-value.
3. Optimize cDNA generation/reverse transcription steps: Then, take an RNA sample with a relatively high amount of target RNA (~107-105 copies/rxn) and run an RT-PCR with a protocol such as this: 10 cycles of 95°C 3sec and a temperature gradient between 60-75 °C for 1 min) and followed by PCR cycles with the best PCR temperature as selected in step 2.
> From this experiment you can select the best temperature/well with the earliest Ct-value.
Further optimizations:
These two steps above combined should already result in a very decent RT-PCR protocol.
Finetuning is possible by shortening the extension-times during PCR phase or further optimizations of the denaturation temperatures (via temperature gradient RT-PCRs e.g. between 85-100°C).
RT-PCR becomes easier once again
zero-step RT-PCRs
Be quick! No isothermal reverse transcription step is needed: A fast start function due to a hotstart aptamer and the engineered, truly thermostable Taq DNA polymerase with reverse transcriptase activity allows immediate cycling.
Even directRT-PCRs are possible like this: Sample extraction and sample lysis is not necessary as immediate hot PCR cycling conditions can brake cell- and virus membranes. Please see our published references to this product.
Superior Performance
Volcano3G is the next generation of our Volcano2G with an improved performance. It offers a variety of applications for a precise and rapid qPCR. It is applicable with common thermal cycling protocols and “zero-step” RT-PCR protocols direct from RNA. As demonstrated here, artificial HPRT1 mRNA was detected in a 10-fold dilution series by using a probe-based qPCR assay and a common thermal RT-PCR protocol.
Improved Sensitivity
Volcano3G shows an improved detection of mRNA down to 500 total copies, which is clearly distinguishable from the No Template Control (NTC). The brighter fluorescent signal provided by the Volcano 3G Master Mix enables an easy and precise quantification. Volcano3G RT-PCR mix showed to be more sensitive than our Volcano2G RT-PCR mix by a factor of 10. Here, artifical HPRT1 mRNA was detected down to 500 copies with Volcano3G RT-PCR 2x Master mix that contains a sensitive DNA binding dye with negligible polymerase inhibition.
Melt your DNA, not your brain
Volcano3G RT-PCR 2x Master mix with GreenDye is suitable for high resolution melting (HRM) experiments, allowing you to quickly identify unspecific amplicons and thus save time and effort when optimizing your PCR.
