RT-real-time-PCR mix with EvaGreen® fluorescent DNA stain
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
Concentration: 2x conc.
Spectroscopic Properties: λexc 500 nm (bound to DNA), λem 530 nm (bound to DNA)
SCRIPT RT-qPCR GreenMaster with highROX is designed for quantitative real-time analyses of RNA templates using the fluorescent DNA stain EvaGreen®. The ready-to-use mix is based on a genetically engineered reverse transcriptase with enhanced thermal stability providing increased specificity, high cDNA yield and improved efficiency for highly structured and long cDNA fragments.
The 2x conc. mix contains all reagents required for RT-qPCR (except template and primers) in one box to ensure fast and easy preparation with a minimum of pipetting steps. The premium quality enzymes and the optimized reaction buffer containing ultrapure dNTPs ensure superior real time PCR results.
RT-qPCR is used to amplify double-stranded DNA from single-stranded RNA templates to allow a rapid real-time quantification of RNA targets. In the reverse transcription step the reverse transcriptase synthesizes single-stranded DNA molecules (cDNA) complementary to the RNA template. In the first cycle of the PCR step the hot-start DNA polymerase synthesizes DNA molecules complementary to the cDNA, thus generating a double-stranded DNA template. The hot-start polymerase activity is blocked at ambient temperature and switched on automatically at the onset of the initial denaturation. The thermal activation prevents the extension of non-specifically annealed primers and primer-dimer formations at low temperatures during PCR setup.
One-step RT-qPCR offers tremendous convenience when applied to analysis of targets from multiple samples of RNA and minimizes the risk of contaminations.
SCRIPT RT-qPCR GreenMaster with highROX (red cap)
Ready-to-use mix of SCRIPT Reverse Transcriptase, Hot Start Polymerase, RNase Inhibitor, dNTPs, reaction buffer, EvaGreen fluorescent DNA stain, ROX and stabilizers.
RNase-free Water (white cap)
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 on the detection instrument.
ROX reference dye:
The SCRIPT RT-qPCR GreenMaster contains ROX passive reference dye. The dye does not take part in the PCR reaction but allows to normalize for non-PCR related signal variation and provides a baseline in multiplex reactions. The reaction chemistry of the kit is optimized for real-time cyclers that are compatible with the evaluation of the ROX reference signal.
Targets can generally be detected from <1 pg to 20 ng poly(A) RNA (mRNA) or 10 pg to 1 μg total RNA. Even lower amounts of RNA may be successfully amplified by using highly expressed transcripts.
Add the following components to a nuclease-free microtube. Pipett on ice and mix the components by pipetting gently up and down. In general, water, RNA and primers should be mixed together before the remaining components are added.
|com-ponent||stock conc.||final conc.||20 μl|
|RNase-free water||-||-||fill up to|
|fill up to|
|RNA template 1)||-||<100 ng||x μl||x μl|
|forward Primer||10 μM||400 nM||0.8 μl||2 μl|
|reverse Primer||10 μM||400 nM||0.8 μl||2 μl|
|SCRIPT RT-qPCR GreenMaster with highROX 2)||2x||1x||10 μl||25 μl|
Continue with reverse transcription and thermal cycling as recommended.
Reverse transcription and thermal cycling:
Place the vials in a PCR cycler and start the following program.
|60-65°C 5)||1 min6)||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 RNA / primer pair.
EvaGreen® is a registered trademark and licensed for sale by Biotium, Inc., Hayward, CA, USA
SYBR® is a registered trademark of Invitrogen Corporation, Carlsbad, California, USA
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