VolcanoCell 2G RT-PCR 2x Master Mix

Volcano Cell 2G RT-PCR 2x Master Mix Direct PCRs

  • This mix has been specifically optimized for direct-from-cell applications.

    Skip the time-consuming and expensive RNA purification. The VolcanoCell2G RT-PCR 2x Master Mix enables you to quantify and detect RNA directly from a cell suspension, or a cell lysate, without a need of purification steps. Simply detach the cells, add the cell suspension directly to your RT-PCR reaction mix, place in your real-time instrument, and start.

    Optionally, use the lysis buffer to lyse the cells directly in the well or flask, simply spin down and add the supernatant directly to your RT-PCR mix.

    The lysates can also be frozen and stored at -20°C.

  • The kit contains an engineered, extremely thermostable reverse transcriptase and combined DNA polymerase, including a specially optimized buffer system that requires only a small amount of cells (100 to 10.000 cells). A separate 50x lysis buffer is included in the kit as well.

    The included Volcano2G DNA polymerase was engineered through multiple generations of directed, artificial evolution and has a half-life at 95°C of >40min. It facilitates a “zero-step” RT-PCR directly from cell suspension or lysate without an isothermal reverse transcription step.

    VolcanoCell2G RT-PCR 2x Master Mix contains all components necessary for a successful and reliable RT-PCR in all standard real-time cycler instruments and is compatible with the common RT-PCR detection methods such as fluorescent realtime PCR dyes or fluorogenic probes.

    Recommended amplicon size should be between 60-300 bp.

    Please note that primers which span an exon-exon junction must be used in order to limit the amplification only to mRNA targets.

    By avoiding expensive, time-consuming cell lysis or RNA purification, VolcanoCell kit is ideal for high-throughput applications, RNAi knockdown or gene expression experiments.The pre-ready 2x mix ensures repeatable RT-PCR results, significantly reduces set-up times and the risk of pipetting mistakes. Only primers and template need to be added. Cell lysates can be easily prepared with the included lysis buffer. However, amplicon size should ideally be between 60-300 bp!

  • 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.

  • RT-PCR activity: A serial dilution of HEK cells in 1x lysis buffer is tested for a successful RT-PCR performance. A 151 bp fragment (HPRT1 mRNA) is amplified in a real-time PCR cycler and visualized as a single amplified product.

    DNA polymerase activity: Volcano3G DNA polymerase 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 info@mypols.de for the lot-specific concentration.

    Lysis buffer: A serial dilution of HEK cells in 1x lysis buffer is tested for a successful RT-PCR performance. A 151 bp fragment (HPRT1 mRNA) is amplified in a real-time PCR cycler and visualized as a single amplified product.

  • VolcanoCell2G publication with more detection examples:

    Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription: a 'zero-step' RT-qPCR protocol.
    “…For a ‘zero-step’ RT-qPCR, cells were lyzed directly in the culture well using VolcanoCell2G Lysis Buffer (myPOLS Biotec) for 15 min at 4 °C. Cell lysate dilutions were prepared in the lysis buffer and stored at −80 °C. Thawed diluted supernatant from approximately 1500 cells was then used as a template for the RT-qPCR reaction. Reaction mixtures (10 µl) contained 5 µl of VolcanoCell2G 2× RT-PCR Master Mix (myPOLS Biotec), 0.1 µM of the respective hydrolysis probe, and 0.4 µM of the respective forward and reverse primers…”
    Chovancova P, Merk V, Marx A, Leist M, Kranaster R.
    Biol Methods Protoc. 2017 Jan;2(1):bpx008.
    Link to publication: https://doi.org/10.1093/biomethods/bpx008

    Find here all myPOLS Biotec references at a glance

Save time with 0-step protocols

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°C of >40 min.

Sensitive RNA detection

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.

Saves time and money

VolcanoCell2G RT-PCR 2x Master mix has a streamlined 'zero-step' workflow that allows you to circumvent laborious purification and extraction steps, saving you both time and money.

Superior sensitivity

VolcanoCell2G RT-PCR 2x achieves detection sensitivities down to single cells. In this example, GAPDH mRNA was detected from a single Lund human mesencephalic (LUHMES) cell following the VolcanoCell2G protocol.

A publication with more examples can be found in the Publications section.

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