» Sign in / Register

SCRIPT RT-qPCR GreenMaster

RT-real-time-PCR mix with EvaGreen® fluorescent DNA stain

Cat. No. Amount Price (EUR) Buy / Note
PCR-514XS 500 μl (2x conc.) 97,38 Add to Basket/Quote Add to Notepad
PCR-514S 2 x 1,25 ml (2x conc.) 389,50 Add to Basket/Quote Add to Notepad
PCR-514L 10 x 1,25 ml (2x conc.) 1.558,00 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

Concentration: 2x conc.

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

Description:
SCRIPT RT-qPCR GreenMaster 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.

Content:

ComponentPCR-514XSPCR-514SPCR-514L
SCRIPT RT-qPCR GreenMaster*
2x conc.
red cap
500 μl2 x 1.25 ml10 x 1.25 ml
RNase-free water
white cap
1.2 ml2 x 1.2 ml2 x 6 ml


*Ready-to-use mix of SCRIPT Reverse Transcriptase, Hot Start Polymerase, RNase Inhibitor, dNTPs, reaction buffer, EvaGreen fluorescent DNA stain and stabilizers.


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.

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.

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-ponentstock conc.final conc.20 μl
assay
50 μl
assay
RNase-free water--fill up to
20 μl
fill up to
50 μl
RNA template 1)-<100 ngx μlx μl
forward Primer10 μM400 nM0.8 μl2 μl
reverse Primer10 μM400 nM0.8 μl2 μl
SCRIPT RT-qPCR Green-Master 2)2x1x10 μl25 μl

1) up to 100 ng polyA RNA or total RNA
2) SCRIPT RT-qPCR GreenMaster 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.

reverse
transcription 3)
50°C10-15 min1x
initial
denaturation 4)
95°C5 min1x
denaturation95°C15 sec35-45x
annealing 5) 55-65°C20 sec35-45x
elongation 6) 72°C30 sec35-45x

3) 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.
4) An initial denaturation time of 5 min is recommended to inactivate the reverse transcriptase
5) The annealing temperature depends on the melting temperature of the primers.
6) 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.

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

Product Citations:
Please click the black arrow on the right to expand the citation list. Click publication title for the full text.