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SCRIPT RT-qPCR SybrMaster

RT-real-time-PCR mix with SYBR® Green fluorescent DNA stain

Cat. No. Amount Price (EUR) Buy / Note
PCR-520XS 500 μl (2x conc.) 97,38 Add to Basket/Quote Add to Notepad
PCR-520S 2 x 1,25 ml (2x conc.) 389,50 Add to Basket/Quote Add to Notepad
PCR-520L 10 x 1,25 ml (2x conc.) 1.558,00 Add to Basket/Quote Add to Notepad

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 494 nm (bound to DNA), λem 521 nm (bound to DNA)

Description:
SCRIPT RT-qPCR SybrMaster is designed for quantitative real-time analyses of RNA templates using the fluorescent DNA stain SYBR® Green. 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.
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-520XSPCR-520SPCR-520L
SCRIPT RT-qPCR SybrMaster*
2x conc.
500 μl2 x 1.25 ml10 x 1.25 ml
PCR-grade water
DNase/RNase/
Phosphatase-free
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, SYBR® Green fluorescent DNA stain and stabilizers.


SYBR® Green fluorescent DNA stain:
SYBR® 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.

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
PCR-grade 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 SybrMaster 2)2x1x10 μl25 μl

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

SYBR® is a registered trademark of Invitrogen Corporation, Carlsbad, California, USA