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TOROGreen® Plant Direct PCR Kit
    Publish time 2019-09-24 16:02    

TOROGreen® Plant Direct PCR Kit is is designed to amplify DNA directly from plant samples. No DNA purification is required prior to PCR. The kit is based on engineered hotstart KOD DNA Polymerase, a extremely robust PCR enzyme,which can tolerant of many PCR inhibitors present in plant material like complex polysaccharides, polyphenols and others. TORO Green® 6×DNA loading buffer contains the fluorescence dye, which is directly detected on 470nm LED transillum inator after the DNA electrophoresis. This kit has been tested with leaves and seeds from a wide variety of plant species. The kit includes a complete set of optimized reagents and detailed protocols making it an ideal choice for amplification of plant DNA.

DESCRIPTION

 TOROGreen® Plant Direct PCR Kit is is designed to amplify DNA directly from plant samples. No DNA purification is required prior to PCR. The kit is based on engineered hotstart KOD DNA Polymerase, a extremely robust PCR enzyme,which can tolerant of many PCR inhibitors present in plant material like complex polysaccharides, polyphenols and others. TORO Green® 6×DNA loading buffer contains the fluorescence dye, which is directly detected on 470nm LED transillum inator after the DNA electrophoresis. This kit has been tested with leaves and seeds from a wide variety of plant species. The kit includes a complete set of optimized reagents and detailed protocols making it an ideal choice for amplification of plant DNA.

FEATURES

-Easy-to-usesample is added directly to PCR reaction, therefore there is no need for time-consuming and expensive DNA purification steps.
-Simple Lysis protocol available—allows more PCR reactions and allows retesting

- High performance: can amplify 40kb genomic DNA and 90%  GC-rich target with 80-fold higher fidelity than Taq DNA polymerase. 

APPLICATION

- Transgene detection

- Genotyping
- Knockout analysis
- Sequencing

COMPONENTS

The kit includes the following reagents, which can be used for 100 reactions for a total 20ul reaction volume.

   Cat No. :DPK-300

 Plant Lysis Buffer                         12ml×1tube 

   2× ASFastTM Direct PCR Mix                               1ml ×1tube

   TOROGreen®  6×DNA loading buffer                1ml ×1tube

 

BRIEF PROCEDURE

PROTOCOL

1. Sample Handling

Direct PCR of plant leaves or seeds 

Leaves: Take a sample from the fresh plant leaves of approximately 0.35-0.5 mm punch disc. Place the disc directly into the PCR reaction (50μL volume). It is recommended to eject the disc into a liquid, rather than onto the wall of an empty tube. Make sure that you see the sample disc in the solution.

Seads:Using a clean scalpel, remove the seed coat and cut a small sample of the seed, Place the sample directly into the PCR reaction (50μL in volume). Note that it is recommended to use dehulled seeds. For very small seeds , use 12 whole seeds and place them directly into the PCR reaction.

 Direct PCR of plant leaves lysis or seeds

a. Thermo lysis protocol

-Take a fresh leaf of approximately 3 mm in diameter or 1-3 seeds and place it in 100μL of Plant Lysis BufferMake sure that you see the sample in the lysis solution.

- Briefly vortex the tube and incubate the sample at 95for 15min in drybath.

- Briefly vortex the tube and centrifuge at 12000rpm for 5 min.

- Use 0.5μL of the supernatant as a template in a 20μL PCR reaction.

b. Homogenize lysis protocol

-Take afresh leaf of approximately 10-15mm in diameter or 5-10 seeds and place it in the tube with zirconia crystal beads.

-Add 600μL of Plant Lysis Buffer into the tube.

-Fully homogenize the sample according the homogenizer instruction book.

- Use 0.5μL of the supernatant as a template in a 20μL PCR reaction.

 

Notes:The Plant Lysis Buffer has been optimized to release DNA from a wide variety of different sample materials such as plant leaves and seeds. This buffer is also suitable for storing the DNA sample up to 1 month at 20. Before storage it is recommended to transfer the supernatant into a new tube. 

2. Standard reaction setup.

1Prepare the mixture on ice as the following components.


 


    Notes:

      -Primers should be 22-35 bases with  Tm 65°C.

      -Optimal primer concentration is 0.3uM. In the case of long targets (10 kb), reduced primers concentration (0.15uM) may give more effective amplification.

      -When PCR yield is low, increased primers concentration (0.5uM) may give more effective amplification.

 3.Set up cycling conditions

 

4.Electrophoresis.

  • Add 1 volume of TOROGreen® 6×DNA loading buffer to 5 volumes of PCR products.

  • Mix well, spin down and load 5ul of the mixture and 2ul of the suitable TOROGreen® Loading Marker.

  • Run on agarose electrophoresis to detect PCR products and marker .No additional dye is required for the PCR products and marker.

STORAGE
     This reagent  his reagent can be stored at 4°C for 1 months. For longer storage,  this reagent should be kept at -20°C 



Ordering Information


Cat.No.


Product Name


Size


Store at


Price


Data Sheet


DPK-300


TOROGreen®Plant Direct PCR Kit 


20μL×100 Reactions

 

-20°C


US$     100.00


 

 

  


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