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Volcano3G® RT-PCR 2x Master Mix (+GreenDye)
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Volcano3G® RT-PCR 2x Master Mix (+GreenDye) is a ready-to-use master mix for reliable qPCR and RT-PCR in all standard PCR cyclers. The mix contains an enzyme blend of a robust Taq DNA polymerase and an engineered, thermostable DNA polymerase with reverse transcriptase activity. It is supplemented with GreenDye (#2000), a highly sensitive DNA binding dye with low polymerase inhibition. It is suitable for high resolution melting experiments.
Volcano3G® RT-PCR 2x Master Mix (+GreenDye) is also available with ROX reference dye (#6401).
We recommend to re-establish the PCR protocol. Primers should be re-designed for higher melting/annealing points (above 65°C) and additionally, RT-PCRs with temperature gradients are performed to establish the best working PCR protocol for your assay with Volcano3G® RT-PCR 2x Master Mix (+GreenDye). Contact us if you need further support.
For PCR detection with probes, we suggest Volcano3G® RT-PCR Probe 2x Master Mix which is optimized for (multiplex) probe assays. It is also available with or without ROX reference (#6101).
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)
- qPCRs
- Melting curve analysis
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How do I have to store the mix?
Keep at -20°C
- 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 simply replace my currently used RT-PCR mix with your Volcano3G® RT-PCR 2x Master Mix (+GreenDye)?
No. We recommend to re-establish the PCR protocol. Primers should be re-designed for higher melting/annealing points (above 65°C) and additionally, RT-PCRs with temperature gradients are performed to establish the best working PCR protocol for your assay with Volcano3G® RT-PCR 2x Master Mix (+GreenDye). Contact us if you need further support. We have already established PCRs for other customers and offer this service for free to you too.
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Volcano3G® RT-PCR 2x Master Mix (+GreenDye) is tested for a successful RT-qPCR performance. A 151 bp fragment (HPRT1 mRNA) is amplified from a RNA dilution series (500.000, 50.000, 5000, 500 copies/rxn) and quantified by the fluorescence of dsDNA-binding GreenDye. The linearity of amplification over the specified serial dilution is demonstrated. Additionally, melting points of the amplification products are analysed. The activity of Volcano3G® DNA polymerase 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 lot-specific concentration. No contamination has been detected in standard test reactions.
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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.
