» Sign in / Register

Direct One-Step RT-qPCR GreenKit

One-Step RT-PCR Kit with EvaGreen® for highly sensitive and specific amplification directly from blood or cell material

Cat. No. Amount Price (EUR) Buy / Note
PCR-519XS 20 reactions x 50 μl 121,98 Add to Basket/Quote Add to Notepad
PCR-519S 100 reactions x 50 μl 487,90 Add to Basket/Quote Add to Notepad
PCR-519L 500 reactions x 50 μl 1.951,60 Add to Basket/Quote Add to Notepad
Excitation (left) and emission (right) spectra of EvaGreen® bound to dsDNA in PBS buffer (pH 7.3).
Excitation (left) and emission (right) spectra of EvaGreen® bound to dsDNA in PBS buffer (pH 7.3).

For in vitro use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles, store dark
stable at 4 °C for up to 4 weeks

Shelf Life: 12 months

Form: liquid

Spectroscopic Properties: λexc 500 nm (bound to DNA), λem 530 nm (bound to DNA)

Description:
Direct One-Step RT-qPCR ProbesKit is designed for quantitative real-time analysis of target RNA directly from whole blood, swabs and animal- or plant tissue without the requirement of any prior RNA purification steps. The kit contains the fluorescent DNA stain EvaGreen®. The fluorescent dye in the reaction mix intercalates into the amplification product during the PCR process and enables the rapid analysis of DNA fragments without the need to synthesize sequence-specific labeled probes.
The enzyme mixture includes a genetically engineered reverse transcriptase and an antibody-inhibited Taq polymerase. The 2x conc. reaction mix contains ultrapure dNTPs and an unique buffer system optimized to resist various PCR inhibitors in unpurified sample material.

The kit ensures fast and easy preparation with a minimum of pipetting steps and is highly recommended for:

  • direct detection of RNA viral pathogens in various tissues
  • direct amplification of target RNA from sample materials
  • point-of-care diagnosis

EvaGreen® Fluorescent DNA Stain:
EvaGreen® Fluorescent DNA Stain is a superior DNA intercalator dye specially developed for DNA analysis applications including real-time PCR (qPCR) and high-resolution DNA melting curve analysis (HRM). Upon binding to DNA, the non-fluorescent dye becomes highly fluorescent while showing no detectable inhibition to the PCR process. The dye is extremely stable both thermally and hydrolytically, providing convenience during routine handling. The high quantum yield, excellent stability and lowest inhibition toward PCR makes it the ideal fluorophore in real-time PCR applications and a superior replacement for the widely used SYBR® Green I dye. To perform the EvaGreen-based assay simply select the optical setting for SYBR® Green or FAM on the detection instrument.

Content:
Direct Enzyme Mix (red cap)
Mix of engeneered reverse transcriptase, antibody-inhibited hot start polymerase and RNase inhibitor in storage buffer with 50 % glycerol

Direct Reaction Mix with EvaGreen (green cap)
2x conc. buffer system containing dNTPs

Extraction Buffer (yellow cap)
10x conc.

PBS (phosphate buffered saline) (blue cap)
10x conc.

PCR-grade Water (white cap)

Sample preparation
1. Whole Blood or Salvia (heparin-, EDTA- or citrate-treated whole blood)

  • Add 1-5 μl of the sample without any pre-treatment directly to the RT-PCR assay.

2. Swab Samples

  • Place the swab brush into a 1.5 ml microcentrifuge tube containing 270 μl PCR-grade Water and 30 μl PBS, 10x conc. (phosphate buffered saline).
  • Rotate the brush 5-10 times.
  • Squeeze the brush and remove it from the tube.
  • Centrifuge at 12,000 g for 3 min at room temperature.
  • Discard the supernatant.
  • Add 90 μl PCR-grade Water and 10 μl Extraction Buffer to the harvested sample.
  • Briefly mix the sample by vortexing and make sure that the sample is soaked with Extraction Buffer.
  • Incubate for 3 min at room temperature for tissue lysis and RNA releasing.
  • Centrifuge briefly and transfer 1-5 μl of the supernatant to the RT-PCR assay.

3. Animal or Plant Tissue

  • Prepare a small piece from animal or plant tissue not exceeding 6 mm in diameter.
  • Crack plant seeds to less than 1 mm in diameter using a BeadBeater, TissueLyser or small hammer.
  • Add Extraction Buffer to the tissue sample as following:


Sample size (diameter)1-2 mm3-4 mm5-6 mm
PCR-grade Water45 μl90 μl135 μl
Extraction Buffer5 μl10 μl15 μl

  • Mix briefly by tapping or vortexing and make sure that the sample is soaked with Extraction Buffer.
  • Incubate for 3 min at room temperature for tissue lysis and RNA releasing.
  • Centrifuge briefly and transfer 1-5 μl of the supernatant to the RT-PCR assay.

Preparation of the RT-PCR Assay
Add the following components to a nuclease-free microtube. Pipett on ice and mix the components by pipetting gently up and down.

componentstock conc.final conc.20 μl assay50 μl assay
Direct Reaction Mix with EvaGreen2x1x10 μl25 μl
Sample--1-2 μl1-5 μl
Forward Primer10 μM400 nM0.8 μl2 μl
Reverse Primer10 μM400 nM0.8 μl2 μl
Direct Enzyme Mix1)25x1x0.8 μl2 μl
PCR-grade water--fill up to
20 μl
fill up to
50 μl

1) Direct Enzyme Mix already contains RNase inhibitor that is recommended and may be essential when working with low amounts of starting RNA.


Reverse transcription and thermal cycling:
Place the vials into a real-time PCR cycler and start the following program.

Reverse
transcription
50 °C30 min1x
Initial
denaturation
95 °C5 min1x
Denaturation95 °C15 sec35-45x
Annealing2)55-65 °C20 sec35-45x
Elongation3)72 °C30 sec35-45x

2) The annealing temperature depends on the melting temperature of the primers.
3) The elongation time depends on the length of the amplicon. A time of 1 min for a fragment of 1,000 bp is recommended.

For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary. Note that optimal reaction times and temperatures should be adjusted for each particular sample/primer pair.