Real-time PCR Master Mix E2

The master mix for RTD-PCR/qPCR contains: EvaGreen, dUTP and UNG

Cat.-no	Description	Amount	Price €	Shop
S160	Real-time PCR Mastermix E2	100 rcs / 50µl	125.00	add

qPCR / Real time PCR Mastermix E2: Datasheet
qPCR / Real time PCR Mastermix E2: Deutsche Beschreibung

Features:
- The Master mix contains dUTP instead of dTTP
- The Master contains UNG (Uracil-N-Glycosylase)
- It contains EvaGreen as fluorescent dye
- The qPCR / RTD-PCR Master mix E2 is ready-to-use and is optimized for high specificity and sensitivity
because of optimized reaction buffer
- easy to use because ready-to-use Master Mix
- The Master Mix can be used with ROX as reference dye (1x concentrated)

Applications:
- Detection and quantification of DNA and cDNA targets
- Profiling gene expression
- Microbial detection
- Viral load determination

Compatibility:

BioRad: iCycler, MyiQ, MiQ 2, iQ 5, CFX-96, CFX-384, MJ Opticon, Option2, Chromo4, MiniOpticon
Qiagen: Roto-Gene Q, Roto-Gene3000, Roto-Gene 6000
Eppendorf: Mastercycler realplex
Illumina: Eco RealTime PCR System
Cepheid: SmartCyler
Roche: LIghtCycler 480, LightCycler 2.0

Description:
The Master Mix contains all reagents required for qPCR (except template and primer) in a premixed 2x concentrated ready-to-use solution. The high specificity and sensitivity of the mix is achieved by an optimized hot-start polymerase. Its 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.
The mix offer dUTP instead of dTTP to prevent carry-over contaminations of DNA from previous PCR reactions. It contains optimized UNG (Uracil-N-Glycosylase) for the treatment at the onset of thermal cycling.

Concentration: The Mastermix is 2x concentrated

List of components qPCR / RTD-PCR Master mix:
Hot-Start Polymerase for qPCR, dATP, dCTP, dGTP, dUTP, EvaGreen, optimized reaction buffer with KCl and MgCl₂, Uracil-N-Glycosylase, stabilizers and enhancers, PCR-grade water

Transportation: with blue ice

Storage: at 4°C for 3 months, at -20°C for more than 12 months, Note: protect from Light

Usage:

Components Volume per reaction final conc.

2X qPCR / RTD-PCR Master mix E2, with
UNG 25 µl 1x

Up-stream primer (10 µM stock) 1,5 µl (range: 0,5-2.5 µl) 300 nM

Down-stream primer (10µM stock) 1,5 µl (range: 0.5-2,5 µl 300 nM

Template DNA 5 µl
(0.1-15 ng/ml plasmid DNA)
(1-10 µg/ml genomic DNA) < 500ng DNA

Sterile dest. Water (included) up to 50 µl total reaction volume

- vortex all solutions carefully before using and before PCR
- may you add the enzyme mix after Template DNA
- an individual optimization of annealing temperature may be necessary for new combinations of primers
and Template DNA

General Thermo-Cycler protocol:

Note: working with EvaGreen just select the optical setting for FAM or SYBR Green at the cycler

Step Time Temperature

UNG treatment 1x2 min 50°C

Initial denaturation 1-3 min 95°C

30-40 Cycles:
Denaturation
Annealing
Extension

15-30 sec
30-65 sec
30 sec
(per 500bp)

95°C
55-65°C
72-75°C

Note:
- an individual optimization of annealing temperature may be necessary for new combinations of primers
and Template DNA

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