One-Step RT-PCR Kit with EvaGreen® for highly sensitive and specific amplification directly from blood or cell material
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
Spectroscopic Properties: λexc 500 nm (bound to DNA), λem 530 nm (bound to DNA)
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:
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.
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)
PBS (phosphate buffered saline) (blue cap)
PCR-grade Water (white cap)
1. Whole Blood or Salvia (heparin-, EDTA- or citrate-treated whole blood)
2. Swab Samples
3. Animal or Plant Tissue
|Sample size (diameter)||1-2 mm||3-4 mm||5-6 mm|
|PCR-grade Water||45 μl||90 μl||135 μl|
|Extraction Buffer||5 μl||10 μl||15 μl|
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.
|component||stock conc.||final conc.||20 μl assay||50 μl assay|
|Direct Reaction Mix with EvaGreen||2x||1x||10 μl||25 μl|
|Sample||-||-||1-2 μl||1-5 μl|
|Forward Primer||10 μM||400 nM||0.8 μl||2 μl|
|Reverse Primer||10 μM||400 nM||0.8 μl||2 μl|
|Direct Enzyme Mix1)||25x||1x||0.8 μl||2 μl|
|PCR-grade water||-||-||fill up to|
|fill up to|
Reverse transcription and thermal cycling:
Place the vials into a real-time PCR cycler and start the following program.
|50 °C||30 min||1x|
|95 °C||5 min||1x|
|Denaturation||95 °C||15 sec||35-45x|
|Annealing2)||55-65 °C||20 sec||35-45x|
|Elongation3)||72 °C||30 sec||35-45x|
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.