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GREEN DYE MASTER MIX/PROBE QPCR MASTER MIX

ORDERING INFORMATION

2x GREEN DYE MASTER MIX (HOT START)

Catalog # Number of 50 l Reactions
Volume ROX (5 M)
Flourescein (500 nM)

Price (EUR)

# S 50
50  
1.25 ml 250 l
100 l    
59,50
# S 200 200  
4 x 1.25 ml 1000 l 400 l    
185,50
# S 400 400  
8 x 1.25 ml 2000 l 800 l    
352,00
# S 2000 2000  
50 ml
10 000 l 4000 l    
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 l Reactions Volume ROX (5 M)
Flourescein (500 nM)

Price (EUR)

# P 50
50  
1.25 ml 250 l
100 l    
51,50
# P 200 200  
4 x 1.25 ml 1000 l 400 l    
161,50
# P 400 400  
8 x 1.25 ml 2000 l 800 l    
306,00
# P 2000 2000  
50 ml
10 000 l 4000 l    
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 dNTPs 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 Tm 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 81.5C. 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 l)

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 60C.

BASIC PROTOCOL FOR 2x PROBE QPCR MASTER MIX

Component

Volume (in l)

Final concentration

2x Master Mix

25

1x

Primer mixture

variable

300 nM

Probes

variable

200 nM

Template DNA

variable

1 10 ng/ 50 l

Distilled, sterile water

Add to a final volume of 50


Recommended melting temperature of primers is 53 60C.


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