NEW

4X 1Step RT qPCR Probe ROX H Kit

Applications


  • Viral RNA detection in diluted low copy number samples

  • Check Application Note for Sars-CoV-2 detection

  • RT qPCR assays based on specific probes: including TaqMan®, Molecular Beacons, Scorpions™ Probes

  • Quantification of any RNA template (mRNA, total RNA, viral RNA), low copy number genes

Benefits


  • Robust 4X qPCR mix for high sample volume input up to 10 µl

  • Detects <5 RNA copies per reaction

  • Successfully tested for Sars-CoV-2 detection

  • Reverse transcription and qPCR in one tube

  • Ideal for multiplex reactions

  • Bulk quantities & custom packaging available

Catalog. # Product Name Size Price Qty
QOP0601 4X 1Step RT qPCR Probe ROX H Kit 200 r of 20 µl
€342.50
QOP0602 4X 1Step RT qPCR Probe ROX H Kit 1000 r of 20 µl
€1,486.50

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Description

4X 1Step RT qPCR Probe Kits are designed for sensitive detection of specific RNAs, including virus RNA, in diluted high-volume samples. They combine a robust 4X qPCR mix (with Hot Start PCR Polymerase) with a 20X blend of thermostable and extremely active Reverse Transcriptase & advanced RNase Inhibitor (RT4 Mix). This formulation allows for a high sample input volume with a reliable outcome of a single step RT qPCR when working with low copy number samples below 5 copies per reaction. 4X 1Step RT qPCR Probe Kits ensure the robust performance of both reverse transcription and qPCR reactions, which allows for the highest sensitivity viral RNA detection under fast qPCR cycling conditions.

The performance of the kits has been tested for Sars-CoV-2 detection according to the recommended Charité Berlin protocol with appropriate primers/probes.

Check Application Note for Sars-CoV-2 detection.

PCR Water supplied in the kit ensures the best performance and reproducibility of the results.

Depending on your instrument requirements, the kit is available as no ROX, ROX L (low) and ROX H (high) versions.

 

Protocols

Print version of the protocol: Product Insert 4X 1Step RT qPCR Probe ROX H Kit
Important Notes

 

  • Take care to prevent RNA from degradation by widely spread and stable RNases.
  • Prepare crude samples, set up reactions in different dedicated areas, use DEPC-treated nuclease-free labware and gloves.
  • Before the cDNA synthesis check RNA quality on denaturing agarose gel to be sure you have good quality material.
  • Use special primer selection programs for good planning.
  • Work with amplicons in a range of 80-200, max 400 bp.
  • Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes, and filter pipet tips.
  • For controls, run reactions in triplets; include a no-template control, no RT Mix control, and positive control in parallel.
  • Thaw and keep reagents on ice. Mix them well before use!
  • Use 2-5 or more microliters of swab extract for 20 μl reaction.
  • 5 minutes are enough for reverse transcription at 45-55°C. For multiplex reactions 10 min RT step might be required.
  • Do not perform annealing/extension for more than 30 seconds. Use 58 °C temperature for this step. Optimization between 58 and 65°C is possible if needed.

 

Prepare a 20 µl reaction:

 

Reverse Primer 0.5 - 1 µM final concentration
Forward Primer 0.5 - 1 µM final concentration
Specific Probe 150 - 500 nM final concentration
1Step RT qPCR Mix, 4X 5 μl
RT4 Mix, 20X 1 μl
Template (extracted RNA
or crude sample from swabs)
2 - 5 μl
(5 - 1x106 copies; you can add up to
7 - 10 μl in case of diluted RNA samples)
PCR Water to 20 μl

 

  • Mix gently, avoid bubbles.
  • Place into the instrument set like:

 

Reverse Transcription 1 cycle: 50°C (45-55°C) - 5 min
Initial denaturation 1 cycle: 95°C - 3 min
Denaturation 50 cycles: 95°C - 15 sec
Annealing/extension 50 cycles: 58°C (58-65°C) - 30 sec

 

  • Follow instrument instructions for melt curve analysis.

Specifications

CAT. SIZE COMPONENTS COMPOSITION

QOP0601

200 r of
20 µl
1 ml - 1Step RT qPCR Probe ROX H Mix, 4X
0.2 ml – RT4 Mix, 20X (RNase Inhibitor+RTase)
1 ml - PCR Water
1Step RT qPCR Probe ROX H Mix, 4X - contains Hot Start Taq, dNTPs, optimized buffer, and high concentration of ROX.
RT4 Mix, 20X (RNase Inhibitor+RTase) - is a 20X concentrated blend of modified MMuLV RT and RNase Inhibitor.

QOP0605

1000 r of
20 µl
5 x 1 ml - 1Step RT qPCR Probe Probe ROX H Mix, 4X
5 x 0.2 ml – RT4 Mix, 20X (RNase Inhibitor+RTase)
5 x 1 ml - PCR Water
1Step RT qPCR Probe ROX H Mix, 4X - contains Hot Start Taq, dNTPs, optimized buffer, and high concentration of ROX.
RT4 Mix, 20X (RNase Inhibitor+RTase) - is a 20X concentrated blend of modified MMuLV RT and RNase Inhibitor.

 

Storage

 

In the dark at -20°C.

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