The RT-qPCR 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
• direct amplification of target RNA from sample
• point-of-care Diagnostics
• Simultanly detection of multiple targets (multiplex PCR)
The kit is recommended for use with Dual Labelled Fluorescent Probes, e.g. TaqMan®, Molecular Beacons or FRET probes but can also be used without fluorescent probes in standard PCR assays. The kit contains an enzyme mixture including 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 impurified sample material.
One.Direct.Step RT-qPCR for Probes 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! Easy protocol.
shipping and storage: transportation with blue ice; storage @ -20°C; for at least 16 months (stable @ +4°C up to 4 weeks), avoid frequently
Manufactured and quality-controlled in accordance with ISO 9001:2000
ProbeMastermix, Direct RT-PCR, SCRIPT PCR Mastermix, Real-time PCR Mastermix for direct reverse transcription from wole blood, nasal swaps
- Direct RT-PCR: Amplification directly from blood or from Nasal or Throat swabs
- The RT-realtime PCR Kit is based on a genetically engineered reverse transcriptase;
- for improved efficiency, thermostability and specivicity; structured and long cDNA fragments;
Final price, free shipping to selected countries3
Extraction Buffer: 1x concentrated
Direct Enzyme: Mix of engineered reverse transcriptase, mAB-inhibited hot start polymerase, dNTPs,
reaction buffers, enhancers and additives
Optional: ROX Dye
Easy Preparation (e.g. 50 µl reaction volume)
1. Whole Blood (not treated heparin-, EDTA- or
citrate-treated whole blood)
• Add 2-5 µl whole blood (for 50 µl reaction volume)
without any pre-treatment directly to the RT-PCR assay.
2. Swab Samples from nasal or swaps
• fill 200 µl Extraction Buffer in a 1,5 ml Tube
• Cut the tip with nasal or throat swap and put to micro
tube and vortex about 15-20 sec
• let absorb and incubate at room temperature for about
• press the tip of the swap to the wall of the microtube
and take it out
• centrifuge the tube extensively and transfer, for a 50µl
reaction volume, 2-5 µl of the supernatant to your RT-
3. Animal or Plant Tissue samples
• Prepare a small piece from animal or plant tissue not
exceeding 8 mm in diameter.
• Crack plant seeds to less than 1 mm in diameter using
a cell-disrupter, Tissue-lyser or small hammer.
• Add 1x Extraction Buffer to the tissue sample using:
1-2 mm - 50 µl
3-4 mm - 100 µl
5-8 mm - 200 µl
• mix Extraction Buffer and sample briefly and
incubate for about 3 min at room temperature
• Centrifuge extensively and and transfer 2-5 µl (for
50 µl reaction volume) of the supernatant to your
Preparation of the RT-PCR Assay
Add the following components to a nuclease-free microtube. Pipette on ice and mix the components by pipetting gently up and down.
|Component||stock conc.||final conc.||20 µl assay||50 µl assay|
|RT-qPCR enzyme mix||2x||1x||10 µl||25 µl|
blood or extracted
|10 µM||300 nM||0,6 µl||1,5 µl|
|10 µM||300 nM||0,6 µl||1,5 µl|
Dual labelled probe / TaqMan Xn 1.)
|10 µl||200 nM||0,4 µl||1 µl|
|optional ROX||25 µM||500nM||0,4 µl||
PCR- grade water
|-||up to 20 µl||
up to 50 µl
1.) for each PCR-Target (Multiplex PCR) take Xn+1 primers and the related amount of TaqMan probe
Note: For each primer on have to optimize the best assay parameters. The optimal primer can vary from 100 - 500 nM.
Reverse transcription and thermal cycling:
Place the vials into a real-time PCR cycler and start the following program.
|reverse transcription||50-55 °C||10-15 min||1x|
|initial denaturation||95 °C||5 min||1x|
|denaturation||95 °C||15 sec||35-45x|
|annealing and elongation||60-65 °C 2)||1 min 3)||35-45x|
Protocol for standard PCR cycler combined with gel - based DNA analysis the following cycling protocol is recommended:
|50 °C||up to 30 min||1x|
|95 °C||3-5 min||
|95 °C||15 sec||35-45x|
|annealing 3.) 4.)||55-65 °C )||1 min 3)||35-45x|
|elongation||67 °C||1 min/kb||35-45x|
|final elongation||67 °C||5 min||1x|
2.) 10 min for amplicons < 200 bp; each 100 bp fragment length need about 3 min longer incubation time
3.)The annealing temperature depends on the melting temperature of the primers.
4.) 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.