Product Datasheet

 
Product Description
 
Product: RealCount One Step RT-qPCR Kit with UNG
Catalog Number: RTQPCR
Product Page: https://www.mol-innov.com/products/realcount-one-step-rt-qpcr-kit-with-ung
Description: The RealCount One Step RT-qPCR Kit with UNG is ideal for accurate quantification of RNA or DNA in a variety of samples such as whole blood, serum, plasma, urine, saliva, cerebral spinal fluid, feces, and plant. The kit can be used for intercalating dye and probe-based chemistries on a variety of qPCR instruments. Aptamer modified warm start Reality Reverse Transcriptase synthesizes complementary DNA (cDNA) from single-stranded RNA or DNA templates. PCR amplification of the cDNA by TaqTivate Hot Start Taq is monitored in real time. The 4X master mix formulation contains inhibitor reducing buffer components optimized for mammalian tissue samples. Uracil-DNA Glycosylase (UNG) and dUTP are included in the master mix to reduce potential false positive results from carry over contamination. The kit contains Reality Reverse Transcriptase (200U/µl), 4X TaqTivate RT-qPCR Master Mix, DTT solution (100mM), 20X TealReveal Dye, and ROX passive fluorescent reference dye (25µM).

Protocol: The following setup is recommended for a 20µl reaction.

Table 1: Single Step Reaction Setup
ComponentVolumeFinal Concentration
TaqTivate Hot Start Master Mix, 4X5µl1X
Reality Reverse Transcriptase, 200U/µl*0.5µl5U/µl
DTT, 100mM*1µl5mM
Target Specific Primers and Probe1µl1X
SampleXµl1pg-100ng
TealReveal Dye, 20X (if required)1µl1X
ROX Reference Dye, 25µM (if required)XµlSee Table 2
Nuclease Free WaterQS to 20µl-

*Reactions must be placed in cycler immediately following completion to maintain the integrity of the Reverse Transcriptase and DTT.

Table 2: Recommended ROX Concentrations
TypeInstrumentROX Final
Concentration
ManufacturerInstrument Name
No ROXRocheLightCycler 480, LightCycler 2.0None
BioRadiCycler, MyiQ, MiQ 2, iQ 5, CFX-96, CFX-384, Chromo4, MJ Opticon, Option2, MiniOpticon
QiagenRoto-Gene Q, Roto-Gene 3000, Roto-Gene 6000
IlluminaEco RealTime PCR System
EppendorfMastercycler realplex
CepheidSmart Cycler
Low ROXABI7500, 7500 Fast30nM
StratageneMX4000P, MX3000P, MX3005P
High ROXABI5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOne plus, Viia7300nM

Table 3: Recommended Cycling Conditions
StageCycling StepCyclesTemperatureHolding Time
1UNG Incubation125°C2min
2RT Incubation155°C15min
3Enzyme Activation195°C2min
4Amplification**4095°C10sec
60°C60sec

**Temperature and holding times will depend on primer set used.

Recommended Storage: -20 C
Shipping Conditions: Cold pack
Source: E. coli
 
Sample Characteristics
 
Lot Number: RTQPCR-SAMPLE
Aliquot: 1 x 200 reactions
Form: Liquid
 
Optimal experimental conditions and working dilutions must be determined by the end user. Sample concentration and volume are for example purposes only and do not necessarily represent the product that will be received. Please contact us for a current lot-specific product datasheet.
 
For in vitro laboratory use only
 
Molecular Innovations, Inc. - 46430 Peary Court, Novi, MI 48377, USA
Tel: (888) 557-5055 - Fax: (248) 896-0148 - info@mol-innov.com