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    Cat. No.
    Size
    List Price*
    QTY
    BIO-65048
    25 Reactions
    $255.00
    +
    BIO-65049
    100 Reactions
    $825.00
    +
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    Description

    An all-in-one-tube mix that delivers ultra-sensitive, highly-specific amplification of a broad range of RNA targets.

    Product Highlights

    • Sensitive – incorporates a blend of high-affinity RT and novel MyTaq HS DNA Polymerase, enabling amplification of low copy number targets from ≥3 pg total RNA
    • Efficient – novel one-step buffer system maximizes the efficiency of both the reverse transcription and PCR steps, delivering improved yield of any target
    • Robust – RT tolerates the higher reaction temperatures required to overcome secondary structure, giving reliable detection of even challenging and GC-rich targets
    • Specific – MyTaq HS DNA Polymerase is an antibody-mediated hot-start enzyme that remains completely inactive during PCR set-up to prevent non-specific amplification
    • Flexible – utilizes gene-specific primers for full-length reverse transcription and subsequent PCR amplification of any RNA target
    • Convenient – an all-in-one-tube mastermix that improves the speed, convenience and accuracy of RT-PCR

    Product Description

    MyTaq™ One-Step RT-PCR Kit has been designed for extremely sensitive and highly reproducible first-stand cDNA synthesis and subsequent PCR in a single tube. The kit contains the latest advances in buffer chemistry, including Bioline ultra-pure dNTPs, together with a proprietary reverse transcriptase and MyTaq HS DNA Polymerase which is our next generation of very high performance, antibody-mediated hot-start DNA polymerase. This ensures that MyTaq One- Step RT-PCR Kit produces fast, highly-specific and ultra-sensitive products for downstream applications.

    MyTaq One-Step Kit consists of reverse transcriptase, 2x MyTaq HS Mix and the potent RNase Inhibitor, RiboSafe, that are blended to create a simple to use all-in-one mix. The kit is ideal for determining the presence or absence of RNA templates and quantifying expression through qualitative, semi-quantitative or quantitative analysis of RNA transcription levels, and the one-step format is also perfect for the synthesis of double stranded cDNA products for subsequent gene expression analysis. The cDNA can be synthesized with starting amounts of RNA template from 3 pg to 1 μg, over a broad temperature range, and up to 50°C to overcome secondary structure and GC-rich sequences, prior to heating to 95°C to inactivate reverse transcriptase and simultaneously to activate the MyTaq™ HS.

    Applications

    • Gene-expression analysis
    • Transcription analysis
    • cDNA cloning
    • Multiplex RT-PCR
    Main
    Highly sensitive, full-length RT-PCR amplification of a broad of RNA targets.

    Introduction to MyTaq


    Overview, features and benefits of the MyTaq product family

    When we compared the performance of our routine supplier’s RTase against Bioline’s MyTaq One-Step RT-PCR, the other supplier’s RTase gave two false negatives in five different grapevine samples tested for Grapevine rupestris stem-pitting-associated Foveavirus. We were convinced to immediately switch.

    University of Adelaide, Australia

    Product Selection

    Please refer to the Real-Time PCR Selection Chart to confirm the recommended product for your PCR application.


    Specification

    Components

    Reagent

    25 Reactions

    100 Reactions

    MyTaq One-Step mix (2x)

    1 x 625 µL

    2 x 1.25 mL

    RiboSafe RNase   Inhibitor (10 u/µL)

    1 x 25 µL

    1 x 100 µL

    Reverse transcriptase

    1 x 12.5 µL

    1 x 50 µL

    DEPC-treated Water

    1 x 1.8 mL

    1 x 1.8 mL

    Storage & Stability

    All components should be stored at -20°C upon receipt for optimum stability. Repeated freeze/thaw cycles should be avoided.

    When stored under the recommended conditions and handled correctly, full activity of the reagents is retained until the expiry date indicated on the outer box label.

    Shipping conditions

    Shipped on dry/blue ice



    FAQs

    In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipetting steps and time and is easy in handling, making it ideal for high throughput screening. In a two-step reaction the reverse transcription reaction and the real-time PCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one real-time PCR reaction and can be archived, eliminating the need to continually isolate RNA. For convenience Bioline sells the SensiFAST cDNA Synthesis Kit separately if you wish to take a two-step approach.

    PCR can be a challenging technique, with various parameters to optimize to achieve the best results. If you are having problems, these could be easily resolved by addressing a few issues. Please see our PCR troubleshooting guide for suggestions and help with your specific problems.

    Observation Recommended Solution(s)
    No or low PCR yield  Enzyme concentration too low – increase the amount of enzyme in 0.5 U increments.
     Primers degraded – check quality and age of the primers.
     Magnesium concentration too low – increase concentration in 0.25 mM increments with a starting  concentration of 1.75 mM.
     Primer concentration not optimized. Titrate primer concentration (0.3-1 µM); ensuring that both  primers have the same concentration.
     Template concentration too low – Increase concentration of template.
     Perform a positive control to ensure that the enzyme, dNTPs and buffers are not degraded and/or contaminated.
    Multiple Bands  Primer annealing temperature too low. Increase annealing temperature. Primer annealing should be at least 5°C below the calculated Tm of primers.
     Prepare master mixes on ice or use a heat-activated polymerase.
     For problems with low specificity. Try adding 3% DMSO (not supplied) to improve specificity.
    Smearing or artifacts  Template concentration too high. Prepare serial dilutions of template.
     Too many cycles. Reduce the cycle number by 3-5 to remove non-specific bands.
     Enzyme concentration too high - decrease the amount of enzyme in 0.5 U increments.
     Extension time too long. Reduce extension time in 0.5-1 minute increments.


    During PCR setup at room temperature, standard polymerases will have some activity. When using a hot-start polymerase, this enzyme will have no activity at room temperature, thus reducing the risk of unspecific products in the reaction due to these mis-primed oligonucleotides. This feature makes this type of polymerase ideally suited to high-throughput applications, where the reactions may be left sitting at room temperature for prolonged periods of time.

    Reviews

    ""When we compared the performance of our routine supplier’s RTase against ..."