qPCR/real-time-PCR Master Mix DLP2

The PCR master mix for qPCR contains: dUTP and UDG (for dual-labeled probes)

Features:
- optimized realtime PCR Mastermix using probe based detection (e.g.
FRET, Molecular Beacons or TaqMan)
- The PCR-Master mix contains dUTP instead of dTTP

- The PCR-Master contains UDG (Uracil-Glycosylase)
- The qPCR / RT-PCR Mastermix DLP1 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 for block based PCR
Cycler
- 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

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 UDG (Uracil-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, optimized reaction buffer with KCl and MgCl₂, Uracil-Glycosylase (UDG), 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

Stability tests / Quality control / Comparison

Manufactured and quality-controlled in accordance with ISO 9001 and 14001

How to use the master mix for realtime PCR.

Components	Volume per reaction	final conc.
2X qPCR Master mix DLP2 with UDG	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:

Step	Time	Temperature
UNG treatment	2 min	50°C
Initial denaturation	2-3 min	95°C
35-50 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

qPCR/real-time-PCR Master Mix DLP2

- optimized realtime PCR Mastermix using probe based detection (e.g.FRET, Molecular Beacons or TaqMan)
- The PCR-Master mix contains dUTP instead of dTTP

- The PCR-Master contains UDG (Uracil-Glycosylase)
- The qPCR / RT-PCR Mastermix DLP1 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 for block based PCR Cycler
- The Master Mix can be used with ROX as reference dye (1x
concentrated)