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PCR Master Mixes

Green Dye / Probe qPCR Master Mixes

GREEN DYE MASTER MIX/PROBE QPCR MASTER MIX

ORDERING INFORMATION

2x GREEN DYE MASTER MIX (HOT START)

Catalog #	Number of 50 µl Reactions	Volume	ROX (5 µM)	Flourescein (500 nM)	Price (EUR)
# S 50	50	1.25 ml	250 µl	100 µl	59,50
# S 200	200	4 x 1.25 ml	1000 µl	400 µl	185,50
# S 400	400	8 x 1.25 ml	2000 µl	800 µl	352,00
# S 2000	2000	50 ml	10 000 µl	4000 µl	1520,00

Please add on your order the type of reference dye needed: ROX or Fluorescein.

2x PROBE QPCR MASTER MIX (HOT START)

Catalog #	Number of 50 µl Reactions	Volume	ROX (5 µM)	Flourescein (500 nM)	Price (EUR)
# P 50	50	1.25 ml	250 µl	100 µl	51,50
# P 200	200	4 x 1.25 ml	1000 µl	400 µl	161,50
# P 400	400	8 x 1.25 ml	2000 µl	800 µl	306,00
# P 2000	2000	50 ml	10 000 µl	4000 µl	1322,00

Please add on your order the type of reference dye needed: ROX or Fluorescein.

DESCRIPTION

The 2x Green DYE Master Mix and the 2x Probe qPCR Master Mix contain all reagents required for Real Time PCR and are designed to make PCR as easy and simple as possible. All components (including Hot Start Taq DNA polymerase) are provided in an optimized concentration. With 2x Green DYE Master Mix all you need to do is to add primers and template DNA, with the 2x Probe qPCR Master Mix addionally probes, thus minimizing the pipetting effort and possible sources of error. ROX or Florescein as reference dye is included in a separate vial.

The Green DYE allows the universal detection of every PCR product. The Green DYE Master Mix and the Probe qPCR Master Mix can be successfully used with every commercial available Real Time PCR instrument.

The highly specific and sensitive Hot Start Polymerase and the extensively purified dNTP´s lead to excellent results of PCR efficiency, correlation coefficient and slope.

Efficiency of the amplification of human GAPDH using log fold serial dilutions from 1 ng to 1 pg on the RG-3000 (Corbett Research).

MELTING CURVE

For Green DYE based amplicon detection, it is important to run a dissociation curve following the real time PCR. This is due to the fact that Green DYE will detect any double stranded DNA including primer dimers, contaminating DNA, and PCR product from misannealed primer.

The T_m of the amplicon correlates with the maximum of the melting curve profile.

Derivative Melting Curve for Standard Curve Samples in Real Time, GAPDH Endogeneous Control

It is apparent that the maximum (melting temperature of the amplicon) occurs at 81.5°C. Also we can see that no contaminating products are present in this reaction. Contaminating DNA or primer dimers would show up as an additional peak separate from the desired amplicon peak

BASIC PROTOCOL FOR 2x GREEN DYE MASTER MIX

Component	Volume (in µl)	Final concentration
2x Master Mix	25	1x
Primer mixture	15	300 nM*
Template DNA	10	100 – 300 nM

* The concentration of primer in the mixture should not exceed 300 nM. Higher concentrations result in unspecific amplification and do not lead to more product. Recommended melting temperature of primers is 53 – 60°C.

BASIC PROTOCOL FOR 2x PROBE QPCR MASTER MIX

Component	Volume (in µl)	Final concentration
2x Master Mix	25	1x
Primer mixture	variable	300 nM
Probes	variable	200 nM
Template DNA	variable	1 – 10 ng/ 50 µl
Distilled, sterile water	Add to a final volume of 50

Recommended melting temperature of primers is 53 – 60°C.

CYCLING PROGRAM FOR 2x GREEN DYE MASTER MIX

Step	Temperature (in °C)	Time	Cycles
Initial activation	95	15 min	1
Denaturation	94	15 sec	45
Annealing	60	30 sec	45
Extension	72	30 sec	45

CYCLING PROGRAM FOR 2x PROBE QPCR MASTER MIX

Step	Temperature (in °C)	Time	Cycles
Initial activation	95	10 min	1
Denaturation	95	15 sec	50
Annealing	60	30 sec	50
Extension	72	30 sec	50

QUALITY CONTROL ASSAYS

FUNCTIONAL ASSAYS	SPECIFICATION
Quantitative PCR
Slope	-3.0 > slope > -3.6
Correlation Coefficient	> 0.990
Efficiency	110% > efficiency > 90%

PHYSICAL ASSAYS	SPECIFICATION
DNA Assay	no contamination
Endonuclease Assay	no activity
DNase Assay	no activity
RNase Assay	no activity

TROUBLE SHOOTING

Observation

Check

Preparing the reaction mix

Mix the master mix a couple of times gently before use in order to avoid a concentration gradient formed in vial.

No vortexing, no centrifugation!

Keep the Master mix protected from light until you use it.

Minimize freeze - thaw cycles. Aliquot the master mix.

Thaw and frozen the reagents by placing them on ice. When thawed, resuspend the samples by gently mixing.

Prepare all solutions in an excess of 5%. First pipette the primer mixture, then add the template and last the Master Mix.

Preventing contamination

Clean lab bench and equipment periodically with 3% hydrogen peroxide.

Wear a clean lab coat and clean gloves. Change gloves whenever you suspect that they are contaminated.

Maintain separate areas and dedicated equipment for: sample preparation, PCR setup, PCR amplification and analysis of PCR products.

Never bring amplified PCR products into the PCR setup area.

Keep reactions and components covered or capped as much as possible.

Use a positive-displacement pipette or aerosol –resistant pipette tips.

Poor correlation

Run the reactions in triplets.

Low quality disposable material

Use only high quality optically clear reaction plates and seals that have been specifically designed for fluorescence applications.

Preparing a plate

Do not use corner wells or use a more robust seal.

Reserve plate positions for positive (control DNA) and negative (water or buffer) controls.

STORAGE

Store at -20°C for 12 months. Multiple freeze-thaw-cycles should be avoided by preparing aliquots.

Rovalab GmbH | Hauffstrasse 38 | 14513 Teltow Germany | Tel. +49 3328 301477 | Fax +49 3328 301478

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Observation	Check

Preparing the reaction mix	Mix the master mix a couple of times gently before use in order to avoid a concentration gradient formed in vial. No vortexing, no centrifugation! Keep the Master mix protected from light until you use it. Minimize freeze - thaw cycles. Aliquot the master mix. Thaw and frozen the reagents by placing them on ice. When thawed, resuspend the samples by gently mixing. Prepare all solutions in an excess of 5%. First pipette the primer mixture, then add the template and last the Master Mix.

Preventing contamination	Clean lab bench and equipment periodically with 3% hydrogen peroxide. Wear a clean lab coat and clean gloves. Change gloves whenever you suspect that they are contaminated. Maintain separate areas and dedicated equipment for: sample preparation, PCR setup, PCR amplification and analysis of PCR products. Never bring amplified PCR products into the PCR setup area. Keep reactions and components covered or capped as much as possible. Use a positive-displacement pipette or aerosol –resistant pipette tips.
Poor correlation	Run the reactions in triplets.
Low quality disposable material	Use only high quality optically clear reaction plates and seals that have been specifically designed for fluorescence applications.
Preparing a plate	Do not use corner wells or use a more robust seal. Reserve plate positions for positive (control DNA) and negative (water or buffer) controls.