DNA / PCR

Plant directPCR 2x Master Mix

 

Description

DNA isolation is not needed anymore - Plant directPCR Mix allows the PCR directly from crude plant samples after a quick lysis step.

The included DNA polymerase was engineered to display a very high resistance against many types of common PCR inhibitors, as well as for robustness and selectivity.

It comes together with an optimized buffer system and a separate lysis buffer.

Plant directPCR 2x Master Mix ensures reproducible results, significantly reduces set-up times and the risk of pipetting mistakes. It can also be used for real-time cycling when adding a suitable real-time dye.

 

ArticleProductSizePrice
#3202S Plant directPCR 2x Master Mix 100 reactions á 25 µl, 1 x 1.25 ml € 50,- 
#3202M Plant directPCR 2x Master Mix 500 reactions á 25 µl, 5 x 1.25 ml € 225,-

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 

 

 

PCR Amplification from different plants

 

A fragment (297 bp) of a highly conserved region of chloroplast DNA from a corn leave (Zea mays),a tomato leaf (Solanum lycopersicum), a soybean (Glycine max),a ficus tree leave (Ficus benjamina), a rape leave (Brassica napus)

and from wall cress (Arabidopsis thaliana) was amplified

after lysing and vortexing a small plant piece for 30 sec

in the provided lysis buffer. The PCR products were

subsequently analysed on a 2.5% agarose gel.

 

Plant direct PCR Mix Arapidopsis Primer

 

Broad Amplification Range

Different-sized amplicons (157 bp; 569 bp; 1326 bp) are selectively amplified from wall cress (Arabidopsis thaliana) leaves using the Plant directPCR 2x Master Mix.

After PCR amplifcation the PCR products were subsequently analysed on a 2.5% agarose gel.

 

Reaction Setup

Reaction Setup

ComponentVolumeFinal concentration
Plant directPCR 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/Sample extract 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 habe a GC-content of 40-60%

Applications

Applications

  • Direct PCR from plants
  • Direct gene detection and quantification
  • Standard PCR
  • Real-time PCR
  • Screening / Hight-throughput PCRs

Typical PCR protocol

Typical PCR protocol

ComponentVolumeFinal concentration
Initial denaturation 95°C 3 min
Denaturation 95°C 10 sec
Annealing* 54-72°C 60 sec/1000 bp   (25-40 cycles)
Hold <10°C  

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

 For a new PCR a temperature gradient to find the optimal temperature is recommended.

 

 

Template preparation

For most fresh plants we recommend, that a 1-4 mm2 part of the leave is diluted in 50 µl of the provided lysis buffer and vortexed for 30 sec to one minute at room temperature. Samples can either be cut with a scalpel or with a puncher. To prevent cross-contamination between samples the scalpel/ puncher must be cleaned properly after each sample.

After the lysis step the lysate should have a light green colour but not be intensively green.

Alternatively, if a centrifuge is available a 1-4 mm2 part of the leave can be diluted in 50 µl of the provided lysis buffer by gently pressing or rubbing the leave against the tube. Before proceeding with the PCR set-up, the lysate should be centrifuged for one minute at 10.000 rpm. The supernatant is directly used in PCR (5-20% of total reaction volume). 

Fresh plants, plant material stored at 4°C or frozen samples are all suitable for the Plant directPCR 2x Master Mix.

 

Real-time PCR

Plant directPCR 2x Master Mix can also be used for real-time PCR, when adding a suitable real-time dye. Please note that high concentrations of  plant sample extracts might quench the fluorescence signal.

 

Quality Control

PCR activity: Plant 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: Plant 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

Plant directPCR 2x Master Mix contains all the components for directPCR from plants, including an engineered DNA polymerase, a specifically developed buffer system, and ultrapure dNTPs. A hot-start formulation of the included DNA polymerase suppresses false amplification. A specifically developed lysis buffer and control primer mixes for plant DNA are also included in the kit.

 

img-faqsFAQ`s

Can you recommend control primers?

Primer control mixes for plant DNA are included in the kit. The mix is 10x ready to use (1 µM each). For the control mix we recommend an extension time of 60 sec and  an annealing temperature of 63°C

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 Plant directPCR kit contains a specially engineered DNA polymerase variant that has a very high resistance against many types of common PCR inhibitors especially those found in plant derived samples. The engineered DNA polymerase features robustness and selectivity and has no exonuclease or endonuclease activity.


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