qPCR Probe 2x Master Mix (LyoCake, freeze-dried)

qPCR Probe 2x LyoCake Master Mix (freeze-dried)

contains all components necessary for a successful and reliable probe based qPCR in all standard realtime PCR cyclers. 

The lyocakes simply need to be rehydrated by adding exactly 218µl of the included Rehydration Buffer to each of the lyocakes, resulting in 250µl of ready-to-use Master Mix.

To the resulting mastermix, only primers, template and a fluorescence-based hydrolysis probe need to be added.

Lyocakes 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 Probe 2x Master Mix ensures reproducible results, significantly reduces set-up times and the risk of pipetting mistakes

Possible applications are:

  • qPCRs
  • Multiplex PCR
  • Realtime PCR
  • Probe-based PCR
  • Pathogene detection
  • Primer extensions
  • Endpoint PCRs
  • Screening / High-throughput PCRs

 

Article Product Size Price
#9801lyoS qPCR Probe 2x LyoCake Master Mix (freeze-dried)

80 reactions à 25 µl, 4 x 0.25 ml

(4x lyocakes)

€ 96,-

#9801lyoM qPCR Probe 2x LyoCake Master Mix (freeze-dried)

400 reactions à 25 µl, 20 x 0.25 ml

(20x lyocakes)

 € 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

Specific quantification over a broad dynamic range.

qPCR Probe PCR Dilution series

 

 

A fragment (92 bp) of the human actin was amplified from a 10-fold dilution series of human genomic DNA extract (100 ng to 1 pg). Hydrolysis probe based realtime PCR was run with a two step protocol on the Lightcycler 96.

Reaction Setup


Reaction Setup

Before use:

Please rehydrate the lyocake by adding exactly 218µl of the respective Rehydration Buffer to each of the PCR mixes, resulting in 250µl of ready-to-use Master Mix. Subsequently invert the closed tube a few times or briefly vortex the mixture before use. The rehydrated 2x PCR Master Mix is now ready to be used for setting up a PCR experiment or can be stored at -20°C.

 

Component Volume Final concentration
qPCR Probe 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)
Probe x µl 0.2 µM (0.05-0.3 µM)
Template** x µl  
Nuclease-free water up to 25µl 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

  • 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 2-step PCR protocol

Step Temperature Time
Initial denaturation 95°C 2 min
Denaturation 95°C 15 sec**
Annealing/Extension* 60°C 60 sec**   (25-40 cycles)

* 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 Probe 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.

A batch specific Certificate of Analysis (CoA) can be provided upon request – please inquire after placing an order.

 

Storage

Costly cooling chains and storage at -20°C are no longer necessary:

Dry qPCR 2x PCR Master Mix lyocakes can be shipped and stored at room-temperature. Once opened, the surrounding air humidity is ideally less than 30%.

The rehydrated 2x PCR Master Mix should be stored 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 Probe 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 Probe 2x Master Mix is also available with ROX (to reach 500nM final concentration in the reaction) as a passive reference dye.

Shipped and stored at room-temperature

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 Probe 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 Probe Master Mix formulated as hot-start?

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


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