Robust real-time RT-PCR master mix with ROX
for highly sensitive and specific amplification directly from tissue, swabs or blood
For general laboratory use.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
stable at 4 °C for up to 4 weeks
Shelf Life: 12 months
SCRIPT Direct RT-qPCR ProbesMaster highROX is designed for quantitative real-time analysis of target RNA directly from animal- or plant tissue, swabs and blood. The mix allows robust amplification avoiding the requirement of any prior RNA purification procedures.
The mix is recommended for use with dual-labeled fluorescent probes, e.g. TaqMan®, Molecular Beacon or Scorpion probes. It provides a powerful tool for multiplex-quantification of sample RNA in a broad dynamic range with exceptional sensitivity and precision.
The mix contains all reagents required for RT-qPCR (except template, primer and labeled fluorescent probe) in a premixed 2 x concentrated ready-to-use solution. High robustness, reliability and sensitivity of the mix are based on a genetically engineered reverse transcriptase and an antibody-blocked hot start polymerase in combination with an optimized and well-balanced buffer system.
The mix ensures fast and easy preparation with a minimum of pipetting steps and is specially recommended for:
ROX Reference Dye
SCRIPT Direct RT-qPCR ProbesMaster highROX contains 500 nM ROX passive reference dye in the final assay. The dye does not take part in the PCR reaction but allows normalization 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.
SCRIPT Direct RT-qPCR ProbesMaster highROX
2x conc. mix of Reverse Transcriptase, antibody-blocked Hot Start polymerase, dNTPs, ROX, reaction buffer, additives and stabilizers
Extraction Buffer (Please handle with care and wear personal protective equipment!)
Before starting, take reagents out from fridge and allow to thaw completely. Vortex all reagents briefly and spin down the liquids.
1. Sample preparation
1.a Blood Samples / Liquid Samples
1.b Samples from nasal or throat swabs
1.c Samples from Animal or Plant Tissue
|Sample size (diameter)||1-2 mm||3-4 mm||5-8 mm|
|1x Extraction Buffer||50 μl||100 μl||200 μl|
2. Preparation of the RT-qPCR Assay
Preparation of a master mix is crucial in quantitative PCR reactions to reduce pipetting errors. Prepare a master mix of all components except template as specified below. A reaction volume of 20-50 μl is recommended for most real-time instruments. Pipet with sterile filter tips and minimize the exposure of the labeled DNA probe to light. Perform the setup in an area separate from DNA preparation or analysis. No-template controls should be included in all amplifications.
|component||stock conc.||final conc.||20 μl|
|SCRIPT Direct RT-qPCR ProbesMaster highROX||2x||1x||10 μl||25 μl|
|Extracted Sample||-||-||1-2 μl||2-5 μl|
|Forward Primer 11)||10 μM||300 nM||0.6 μl||1.5 μl|
|Reverse Primer 11)||10 μM||300 nM||0.6 μl||1.5 μl|
|TaqMan® / Dual Labeled Probe 11)||10 μM||200 nM||0.4 μl||1 μl|
|Forward Primer 22)||10 μM||300 nM||0.6 μl||1.5 μl|
|Reverse Primer 22)||10 μM||300 nM||0.6 μl||1.5 μl|
|TaqMan® / Dual Labeled Probe 22)||10 μM||200 nM||0.4 μl||1 μl|
|PCR-grade water||-||-||fill up to|
|fill up to|
Mix the tubes briefly and spin down to remove bubbles.
3. RT-PCR Cycling
Switch on the real-time PCR cycler and set all cycling parameters as recommended in the table below. Place the vials into the instrument and start the program.
|50-55 °C||10-15 min||1x|
|95 °C||5 min||1x|
Annealing and elongation
60-65 °C 4)
To obtain optimal specificity and amplification results an individual optimization of the recommended parameters is recommended for each particular sample/primer pair.
4. Data Analysis
BIOZ Product Citations: