DNA / PCR

Allstar directPCR 2x Master Mix

 

Description

DNA isolation is not needed anymore - Allstar direct PCR Mix allows the PCR directly from crude samples such as blood, saliva or plants or after a quick lysis step.

 

Allstar DNA polymerase has been specifically engineered and selected to display a very high resistance against many types of common PCR inhibitors. The engineered DNA polymerase features robustness and selectivity. It comes together with an optimized buffer system and a separate lysis buffer.

 

The pre-ready 2x Master Mix ensures repeatable results, significantly reduces set-up times and the risk of pipetting mistakes.

 

ArticleProductSizePrice
#3001SAllstar directPCR 2x Master Mix100 reactions á 25 µl, 1 x 1.25 ml € 55,-
#3001MAllstar directPCR 2x Master Mix500 reactions á 25 µl, 5 x 1.25 ml€ 250,-

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Example Results

Example Results

Abbildung Allstar2

PCR Amplification from human specimen

 

A fragment (237 bp) of the highly conserved non-coding region upstream of the SOX21 gene was amplified from saliva, blood (citrate or EDTA treated) either directly or after a quick lysis step. As positive control 10 ng of a human genomic DNA extract were used, as negative control H2O. PCR products were subsequently analysed on a 2.5% agarose gel.

 

 

 

 

 

Abbildung Allstar1

PCR Amplification from plants

 

 

A fragment (297 bp) of a highly conserved region of chloroplast DNA from a potato leave (Solanum tuberosum), soybean (Glycine max), tomato leaf (Solanum lycopersicum), and from a ficus tree leave (Ficus benjamina) was amplified after lysing and vortexing a small plant piece for 30 sec in the separately provided lysis buffer. PCR products were subsequently analysed on a 2.5% agarose gel.

 

Reaction Setup

Reaction Setup

ComponentVolumeFinal concentration
Allstar directPCR 2x Master Mix12.5  µl1x
Primer forward (10 µM)*0.5 µl0.2 µM (0.05-1 µM)
Primer reserve (10 µM)*0.5 µl0.2 µM (0.05-1 µM)
Template/Sample extractx    µl 
Nuclease-free waterup to 25 µl total reaction volume 

Keep all components on ice.
Spin down and mix all solutions carefully before use.
* Primers should ideally habe a GC-content of 40-60%

Applications

Applications

  • Direct PCR from plants and mammalian specimen
  • Direct gene detection and quantification
  • Standard PCR
  • Realtime PCR
  • Screening / Hight-throughput PCRs

Typical PCR protocol

Typical PCR protocol

StepTemperatureTime
Initial denaturation95°C2 min
Denaturation95°C15 sec
Annealing*54-72°C30 sec     (25-40 cycles)
Extension72 °C2 min / 1000 bp
Hold<10°Chold

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

 

 

Template preparation

Saliva

Fresh saliva should be collected in a reaction tube and centrifuged in a standard benchtop centrifuge for 20s at maximum speed. 2-5% of the supernatant can directly be used in PCR without further treatment.

Alternatively saliva can be treated with a 5x excess of lysis buffer. After vortexing, the reaction is cleared by centrifugation in a standard benchtop centrifuge for 60s at maximum speed. The supernatant is directly used in PCR (2-5% of total reaction volume).

 

Blood

We recommend using 1-5% of blood (treated or untreated) directly in PCR with out further treatment.

Alternatively the blood sample (treated or untreated) can be diluted with a 2-5 fold excess of lysis buffer and vortexed for one minute. The supernatant is directly used in PCR (1-5% of total reaction volume).

Please notice that the blood sample will quench the fluorescence of a realtime dye. Please check our recommendations under Realtime PCR.

 

Plants

 For plants we recommend, that a 1-4 mm2 part of the leave is diluted with 50 µl of lysis buffer and vortexed for 30 sec. The supernatant is directly used in PCR (1-5% of total reaction volume). Alternatively an approximately 1 mm2 part of a leave can be directly used for PCR Also other parts of plants might be used as templates directly or after lysis.

 

Realtime PCR

Allstar directPCR 2x Master Mix can also be used for realtime PCR, when adding a suitable realtime dye. Please note that high concentrations of sample extracts or blood might quench the fluorescence signal.

For blood: Try increased concentrations of realtime dye or pipette the blood sample directly in the PCR tube and let it dry at room temperature for approx. 1/2 h before you add the remaining PCR components.

 

Quality Control

PCR activity: Allstar directPCR 2x Master Mix was tested for successful PCR performance. A 297 bp fragment (chloroplast) was amplified from a plant leave and analyzed by agarose gel electrophoresis.

DNA polymerase activity: Allstar 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

Allstar directPCR 2x Master Mix is containing all the components necessary for PCR, including an optimized reaction buffer, ultrapure dNTPs and an engineered DNA polymerase.

 

Dilution/Lysis buffer is provided separately.

 

Control primer mixes for mammalian and plant DNA.

 

Allstar-direct_teaser

 

img-faqsFAQ`s

Can you recommend control primers?

Primer control mixes for mammalian DNA and plant DNA are included in the kit. Both mixes are 10x ready to use (1 µM each). For both control mixes we recommend an extension time of 45 sec. For the plant control reaction we recommend an annealing temperature of 60°C for the mammalian control reaction an annealing temperature of 66°C.

What is the sequence of the mammalian control primers?

The control primer mix for mammalians included in the kit amplifies a 237 bp fragment of highly conserved non-coding region upstream of the SOX21 gene. The primer mix contains degenerate primers and can be used with wide range of vertebrate species. Primer sequences are:

Primer Mammalian 1 (24 nt)
: 5’-AGCCCTTGGGGASTTGAATTGCTG -3’

Primer Mammalian 2 (27 nt): 5’-GCACTCCAGAGGACAGCRGTGTCAATA -3’

You can find more information in this reference:

Highly Conserved Non-Coding Sequences Are Associated with Vertebrate Development. PLoS. Biol. 2005; 3: e7. A. Woolfe, M. Goodson, D. K. Goode, P. Snell, G. K. McEwen, T. Vavouri, S. F. Smith, P. North, H. Callaway, K. Kelly, K. Walter, I. Abnizova, W. Gilks, Y. J. K. Edwards, J. E. Cooke, and G. Elgar

What is the sequence of the plant specific control primers?

The plant specific control primer mix included in the kit amplifies a 297 bp fragment of a highly conserved region of chloroplast DNA. The primer mix can be used with a large number of plant species. Primer sequences are:

Primer Plant 1 (20 nt)
: 5’-AGTTCGAGCCTGATTATCCC -3’

Primer Plant 2 (20 nt)
: 5’-GCATGCCGCCAGCGTTCATC -3’

You can find more information in this reference:

A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol. Ecol. 1995; 4: 129–131. B. Demesure, N. Sodzi, and R. J. Petit

What kind of DNA polymerase is included in the kit?

The Allstar directPCR kit contains a Taq DNA polymerase variant that has a very high resistance against many types of common PCR inhibitors. The engineered DNA polymerase features robustness and selectivity and has no exonuclease or endonuclease activity.


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