DNA / PCR

qPCR GreenDye 2x Master Mix

 

is a ready to use reaction mix. It contains all components necessary for a successful and reliable qPCR in all standard realtime PCR cyclers. Only primers and template need to be added.

This mix contains an engineered DNA polymerase and provides robust PCR performance for a wide range of qPCR applications. The buffer is optimized to function with a wide range of templates including human-, mammal-, and plant-derived samples. The qPCR GreenDye 2x Master Mix ensures reproducible results, significantly reduces set-up times and the risk of pipetting mistakes. qPCR GreenDye 2x Master Mix is available with or without ROX as a passive reference dye.

 

ArticleProductSizePrice
#9601S qPCR GreenDye 2x Master Mix 100 reactions á 25 µl, 1 x 1.25 ml € 65,-
#9601M qPCR GreenDye 2x Master Mix 500 reactions á 25 µl, 5 x 1.25 ml € 300,-
#9701S qPCR GreenDye 2x Master Mix (+ROX) 100 reactions á 25 µl, 1 x 1.25 ml € 65,-
#9701M qPCR GreenDye 2x Master Mix (+ROX) 500 reactions á 25 µl, 5 x 1.25 ml € 300,-

Bulk offer: Should you require larger amounts (> 10 ml), significant discounts are available. Please contact us to receive an individual quotation.

 

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Example Results

Example Results

Specific, linear quantification over a broad dynamic range.

qPCR GreenDye PCR Dilution series

A fragment (92 bp) of the human actin was amplified from a 10-fold dilution series (100 ng - 1 pg) of human genomic DNA extract. Reactions were run on the LightCycler 96 with a three-step PCR protocol.

 

 

 

 

qPCR GreenDye melting curvesA single melting peak demonstrates the uniformity of the PCR product.

Reaction Setup

Reaction Setup

ComponentVolumeFinal concentration
qPCR GreenDye 2x Master Mix 12.5 µl 1x
Primer forward (10 µM)* 0.5 µl 0.2 µM (0.05-1 µM)
Primer reverse (10 µM)* 0.5 µl 0.2 µM (0.05-1 µM)
Template** x µl  
Nuclease-free water up to 25 µl total reaction volume  

Keep all components on ice.
Spin down and mix all solutions carefully before use.
* Primers should ideally have a GC content of 40-60%. For optimal results we recommend amplicon length in the range of 60 to 300 bp.
**Suggested template concentration should be about 1 ng - 300 ng (genomic DNA) or 1 ng – 1 pg (plasmid/viral DNA). 

Recommendations

Recommendations for sample handling

  • The realtime dye is light sensitive: exposure should be minimized.
  • Thaw and keep all reagents on ice.
  • Spin down and mix all solutions carefully before use.
  • Always include a control without template.
  • Primers should ideally have a GC content of 40-60%. For optimal results we recommend amplicon lengths in the range of 60 to 300 bp.
  • Minimize the number of freeze-thaw cycles by storing the product in aliquots. For a day-to-day use, we recommend keeping an aliquot at 4°C.
  • We recommend the use of disposable gloves, DNase and RNase free filter tips and plastics.

qPCR protocol

Typical 3-step PCR protocol

StepTemperatureTime
Initial denaturation 95°C 2 min
Denaturation 95°C 15 sec**
Annealing* 50-72°C 30 sec**    
Extension 72 °C 30 sec**     (25-40 cycles)
Optional melting step    

* Typically, the annealing temperature is about 3-5°C below the calculated melting temperature of the primers used.

** Suggested cycling times depend strongly on the cycler, template and amplicon length.

 

 

Quality Control

qPCR GreenDye 2x Master Mix is tested functionally using qPCR. The product demonstrates linearity of amplification over a specified serial dilution of human genomic DNA.

DNA polymerase activity: DNA polymerase activity has been monitored and adjusted to a specific DNA polymerase activity using an artificial DNA template and a DNA primer.

Enzyme-concentration has been determined by protein-specific staining. Please inquire more information at info@mypols.de for the lot-specific concentration.

No contamination has been detected in standard test reactions.

 

Storage

This product is shipped on cool packs. Please store the product upon arrival at -20°C.
Minimize the number of freeze-thaw cycles by storing in aliquots. For a day-to-day use, we recommend keeping an aliquot at 4°C.

 

Composition / Content of the Product

qPCR GreenDye 2x Master Mix contains all components necessary for rapid, sensitive and reproducible quantification of DNA and cDNA. An engineered DNA polymerase, an optimized buffer including ultrapure dNTPs and a realtime dye are key components of the ready to use mix. A hot-start formulation of the included DNA polymerase prevents false amplification during the reaction set-up. Optionally our qPCR GreenDye 2x Master Mix is also availible with ROX (to reach 500nM final concentration in the reaction) as a passive reference dye.

 

qPCR_teaser

 

img-faqsFAQ`s

How can I optimize the PCR reaction conditions?

1. The annealing temperature can usually be optimized. Try a temperature gradient and determine the best annealing temperature, which yields in the cleanest product.

2. Try to shorten the extension and annealing time. Too long and too many cycles may lead to over-amplification and side-products.

 

 

Why is the qPCR GreenDye 2x Master Mix available with or without ROX?

 

ROX is a passive reference dye that is needed by some types of qPCR cyclers. If you are not sure whether you need ROX in your mix or not please have a look in the users handbook of your qPCR cycler.

 

Is the qPCR GreenDye 2x Master Mix formulated as hot-start?

 

Yes! The DNA polymerase included in the qPCR GreenDye 2x Master Mix is hot-start formulated. This prevents false amplification during the reaction set-up.


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