One-Step RT-qPCR Kit

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Catalogue No: abx460027
Price: US$304.50
(Size: 100 rxns)

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Datasheet SDS
Abbexa's One-Step RT-qPCR Kit provides high sensitivity of the target RNA level due to its reverse transcriptase and RNase inhibitors mix which aim to diminish RNA degradation and mispriming during reaction setup and reverse transcription to guarantee optimal RT efficiency. The kit also contains a 2X concentrated ready-to-use universal qPCR Master Mix, optimized for probe-based real-time PCR and compatible with the majority of commercially available real-time PCR systems (ROX-independent and ROX-dependent). It contains antibody-mediated hot-start Taq DNA polymerase, dNTPs, MgCl2, enhancers, stabilizers and essentials for a successful PCR reaction.

Kit Components (100 rxns):
  • 2X Universal qPCR Master Mix: 1 ml
  • Enzyme Mix: 20 µl


Target One-Step RT-qPCR Kit
Tested Applications RT-PCR
Storage Store at 4 °C for up to 3 months. For long-term storage, store at -20 °C. Avoid repeated freeze/thaw cycles.
Buffer Contains 30-50 mM Tris-HCl, 0.3-0.5 mg/ml BSA and < 0.3% Triton X-100.
Availability Shipped within 10-15 working days.
Note This product is for research use only.
Directions for use Assay Procedure:

  1. Bring all reaction components to 4 °C and ensure all vials are thawed. Briefly centrifuge the vials before opening to ensure complete recovery of vial contents. Avoid exposure to light.
  2. Add components to a PCR tube on ice or at room temperature according to the table below. Adjust the volumes accordingly for different reaction volumes. The concentration of primers may require optimization.

    Component Volume
    2X Reaction Mix10 µl
    Enzyme Mix0.2 µl
    Forward and Reverse PrimersVariable
    Recommended Final Concentration: 300 nM each
    Fluorogenic Probe(s)Variable
    Recommended Final Concentration: 150-250 nM each
    RNA Template Variable
    Total RNA: 1 ng - 5 µg
    Nuclease-Free Water Variable, up to 20 µl
    Total Reaction Volume 20 µl

  3. Mix by gently pipetting up and down.
  4. Close the lid on the tube and vortex for at least 30 seconds.
  5. Briefly centrifuge the tubes to remove any air bubbles.
  6. Set up the thermal cycling conditions according to the table below. Optimization may be required. Load the PCR tubes into the PCR instrument and commence the run.

    Number of Cycles Stage Temperature Time per Cycle
    1 cycle cDNA Synthesis 42 °C 15 min
    1 cycle Pre-Denaturation 95 °C 5 min
    35-45 cyclesDenaturation95 °C 10 seconds
    Annealing60 °C60 seconds
    1 cycle Instrument Cooling40 °C10 seconds

  7. Perform data analysis according to the instrument's manual.

Notes:
  • Avoid cross-contamination with DNA by cleaning workstation areas with 10% bleach rather than ethanol, as this will hydrolyze and dissolve any residual DNA.
  • Poor or no signal may be observed if inhibitors are present, the template has degraded, or the thermal cycling extension time is insufficient.
  • High primer concentrations may result in primer dimers.
  • A thermal gradient can be carried out to determine the optimal thermal cycling conditions for a specific primer set.
Research Articles on One-Step RT-qPCR Kit


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