DNA / PCR

qPCR GreenDye UDG 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 provides robust PCR performance for a wide range of qPCR applications with a wide range of templates including human-, mammal-, and plant-derived samples. This 2x Master Mix ensures reproducible results, significantly reduces set-up times and the risk of pipetting mistakes. UDG and dUTP prevent reamplification of carryover PCR products between reactions.

 

ArticleProductSizePrice
#8901 S qPCR GreenDye UDG 2x Master Mix 100 reactions à 25 µl, 1 x 1.25 ml € 85,-
#8901 M qPCR GreenDye UDG 2x Master Mix 500 reactions à 25 µl, 5 x 1.25 ml € 400,-

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

Faster Detection and Higher Sensitivity

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

 

 

 

 

Faster Detection and Higher Sensitivity

 

 

An uracil-containing amplicon was used as template using the qPCR GreenDye UDG 2x Master Mix (red curves).  As positive controls UDG was heat-inactivated previous to the PCR by incubation at 95°C for 2 min (blue curves, three template dilutions). Reactions were run on the LightCycler 96 with a three-step PCR protocol.

 

Reaction Setup

Reaction Setup

ComponentVolumeFinal concentration
qPCR GreenDye UDG 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
UDG incubation 50°C 2-5 min
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 UDG 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 UDG 2x Master Mix contains all components necessary for rapid, sensitive and reproducible quantification of DNA and cDNA with integrated UDG carryover prevention technology. An engineered DNA polymerase, an optimized buffer including ultrapure dNTPs (including dUTP instead of dTTP), a realtime dye and UDG 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.

 

qPCR_teaser

 

img-faqsFAQ`s

 

How does the UDG and dUTP carryover prevetion technology work?

 

qPCR GreenDye UDG 2x Master Mix contains UDG and dUTP instead of dTTP to prevent the reamplification of carryover PCR products between reactions. dUTP ensures that any amplified DNA will contain uracil, while UDG removes uracil residues from single- or double-stranded DNA. A UDG incubation step before PCR cycling cleaves any contaminating dU-containing product from previous reactions. UDG is then inactivated by the high temperatures during normal PCR cycling, thereby allowing the amplification of target sequences.

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.

 

 

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

 

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


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