Robust real-time RT-PCR master mix with SYBR Green and ROX
for highly sensitive and specific amplification directly from tissue, swabs or whole blood
For in vitro use only!
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
SCRIPT Direct RT-qPCR SybrMaster highROX is designed for quantitative real-time analysis of target RNA directly from animal- or plant tissue, swabs and whole blood. The mix allows robust amplification avoiding the requirement of any prior RNA purification procedures.
The mix contains SYBR® Green Fluorescent DNA Stain. It allows fast and easy quantification of sample RNA over a wide dynamic range with exceptional sensitivity and precision.
The mix contains all reagents required for RT-qPCR (except template and primers) 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
The SCRIPT Direct RT-qPCR SybrMaster 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 variations in instruments that are compatible with the evaluation of the ROX reference signal.
SCRIPT Direct RT-qPCR SybrMaster highROX
2 x conc. mix of Reverse Transcriptase, antibody-blocked Hot Start polymerase, dNTPs, SYBR® Green DNA intercalator dye, ROX, reaction buffer, additives and stabilizer
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 Whole Blood (not recommended for heparin-, EDTA- or citrate-treated whole blood)
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|
|PCR-grade water||45 μl||90 μl||180 μl|
|Extraction Buffer||5 μl||10 μl||20 μl|
2. Preparation of the PCR 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 SybrMaster||2x||1x||10 μl||25 μl|
|Extracted Sample or whole blood||-||-||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|
|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|
60-65 °C 3)
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