Robust real-time RT-PCR master mix with UNG
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
stable at 4 °C for up to 4 weeks
Shelf Life: 12 months
SCRIPT Direct RT-qPCR ProbesMaster UNG 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 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:
The mix contains UNG (Uracil-N-Glycosylase) and dUTP instead of dTTP to eliminate carry-over contamination of DNA from previous PCR reactions. The UNG treatment at the onset of thermal cycling removes uracil residues from dU-containing DNA and prevents it from serving as template.
ROX Reference Dye
The mix can also be used in combination with ROX reference dye (#PCR-351) in PCR instruments that are compatible with the evaluation of the ROX signal.
SCRIPT Direct RT-qPCR ProbesMaster UNG
2x conc. mix of Reverse Transcriptase, antibody-blocked Hot Start polymerase, UNG, dNTPs, 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 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 ProbesMaster UNG||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|
|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|
|ROX Reference Dye|
|25 μM||500 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 5)
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