SCRIPT RT-qPCR GreenMaster highROX

For general laboratory use.

Shipping: shipped on gel packs

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

Concentration: 2x conc.

Spectroscopic Properties: λ_exc 495 nm, λ_em 520 nm (dye bound to DNA)

Description:
SCRIPT RT-qPCR GreenMaster highROX is designed for quantitative real-time analyses of RNA templates using a green-fluorescent DNA stain that is structurally similar to SYBR® GREEN and has been specially developed for DNA analysis applications including real-time PCR (qPCR). 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) allow fast and easy preparation with a minimum of pipetting steps. The premium quality enzymes and the optimized reaction buffer ensure superior real time PCR results.
SCRIPT RT-qPCR GreenMaster is used to amplify double-stranded DNA from single-stranded RNA templates. In the RT step the reverse transcriptase synthesizes single-stranded DNA molecules (cDNA) complementary to the RNA template. In the first cycle of the PCR, the Hot Start Taq polymerase synthesizes DNA molecules that are complementary to the cDNA, thus generating a double-stranded DNA template. In the following cycle rounds, the DNA polymerase amplifies this double-stranded DNA template exponentially.
In one-step RT-qPCR, all components for reverse transcription and PCR are combined in one tube so that both reactions take place one after the other without opening the tube. This offers enormous convenience when analyzing targets from multiple RNA samples and minimizes the risk of contamination.
The master mix already contains an optimized amount of RNase inhibitor to prevent a decrease in sensitivity due to the degradation of RNA by RNase contamination when using small amounts of templete material.

ROX reference dye:
The SCRIPT RT-qPCR GreenMaster highROX contains 500 nM ROX passive reference dye in the final assay. 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.

Content:
SCRIPT RT-qPCR GreenMaster highROX
Ready-to-use mix of SCRIPT Reverse Transcriptase, Hot Start Polymerase, RNase Inhibitor, dNTPs, reaction buffer, green-fluorescent DNA stain, ROX and stabilizers.

PCR-grade Water

Green-fluorescent DNA stain:
Green-fluorescent DNA stain is a superior DNA intercalator dye specially developed for DNA analysis applications like real-time PCR (qPCR). Upon binding to DNA, the non-fluorescent dye becomes highly fluorescent while showing only lowest inhibition to the PCR process. The dye is stable both thermally and hydrolytically, providing convenience during routine handling.

Sensitivity:
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.

Preparation of the RT-qPCR assay
Add the following components to a nuclease-free microtube and mix the components by pipetting gently up and down. In general, water, RNA and primers should be mixed together before adding the master mix.

¹⁾ up to 100 ng polyA RNA or total RNA
²⁾The optimal concentration for primers and probe may vary from100 to 500 nm.
⁴⁾ The Mix already contains RNase inhibitor that may be essential when working with low amounts of starting RNA.

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. Select SYBR® GREEN as fluorescence dye and collect the signal in the corresponding channel.

⁵⁾ A reverse transcription time of 10 min is recommended for optimal amplicon lengths between 100 and 200 bp. Longer amplicons up to 500 bp may require a prolonged incubation of 15 min. Add 3 min for each additional 100 bp. The optimal temperature depends on the structural features of the RNA. Increase the temperature to 55°C for difficult templates with high secondary structure. Note that optimal reaction time and temperature should be adjusted for each particular RNA.
⁶⁾ An initial denaturation time of 5 min is recommended to inactivate the reverse transcriptase
⁷⁾ The annealing temperature depends on the melting temperature of the primers.
⁸⁾ 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 RNA / primer pair.

® SYBR is a trademark of Molecular Probes, Inc.

BIOZ Product Citations: