Whole Genome Amplification

Plant Tissues


Extracting DNA from plant tissue is a complicated process due to the tough cell wall that surrounds most plant cells. Genomic DNA from plant material can be damaged during the extraction process, resulting in low yields of high quality genomic material. As a result, the researcher’s ability to perform downstream analysis is challenged. GenomePlex® Whole Genome Amplification has been used to amplify genomic DNA from soybean, corn, tomato, purple coneflower, and ginseng.

Required Products

  • GenElute™ Plant Genomic DNA Miniprep Kit (Product No. G2N10)

 

Materials to be Supplied by the User

  • Plant samples
  • 1.5 ml microcentrifuge tubes (Product No. T9661)
  • Ethanol (Product No. E7023)
  • Microcentrifuge (with rotor for 2 ml tubes)
  • Water, molecular biology reagent (Product No. W4502)
  • 55 °C water bath or heat block

 

Extraction of DNA from Plant Tissues
It is recommended to use the Sigma GenElute Plant Genomic DNA Miniprep Kit (Product No. G2N10) for this procedure.

  1. Grind approximately 50 mg leaf punch into a fine powder with liquid nitrogen. Keep the sample on ice for immediate use or freeze at –70 °C.
  2. Add 350 µl of Lysis Solution (Part A) and 50 µl of Lysis Solution (Part B) and thoroughly mix by vortexing. A white precipitate will form upon the addition of Lysis Solution Part B.
  3. Incubate the mixture at 65 °C for 10 minutes with occasional inversion to dissolve the precipitate.
  4. Add 130 µl of Precipitation Solution, mix by inversion, and place the sample on ice for 5 minutes.
  5. Centrifuge at maximum speed (12,000–16,000 × g) for 5 minutes to pellet the cellular debris, proteins and polysaccharides.
  6. Carefully pipette the supernatant onto a GenElute Filtration Column (blue insert with a 2 ml collection tube).
  7. Centrifuge at maximum speed for 1 minute. Discard the Filtration Column and retain the collection tube.
  8. Add 700 µl of Binding Solution directly to the flowthrough (liquid from step 7). Mix thoroughly by inversion.
  9. Insert the GenElute Miniprep Binding Column (red O-ring) into the provided microcentrifuge tube.
  10. Add 500 µl of the Column Preparation Solution to each Miniprep Column and centrifuge at
    12,000 × g for 1 minute. Discard the flow-through liquid.
  11. Pipette 700 µl flow-through (from step 8) onto the Miniprep Column prepared in the previous step.
  12. Centrifuge at maximum speed for 1 minute and discard the flow-through.
  13. Apply the remaining lysate (from step 8) and repeat centrifugation for 1 minute at maximum speed and discard the flow-through.
  14. Place the Binding Column in a fresh 2 ml collection tube and apply 500 µl diluted Wash Solution to the column (be sure to add ethanol to the Wash Solution Concentrate prior to first time use).
  15. Centrifuge at maximum speed for 1 minute. Discard flow-through and retain the collection tube.
  16. Add another 500 µl of diluted Wash Solution to the column and centrifuge at maximum speed for 3 minutes to dry the column.
  17. Transfer the binding column to a fresh 2 ml collection tube.
  18. Apply 100 µl of pre-warmed (65 °C) Elution Solution to the column and centrifuge at maximum speed for 1 minute. Repeat the elution.
  19. Store the eluted DNA at –20 °C or proceed to the amplification step.

 

Application Data

GenomePlex Whole Genome Amplification Performed on Plant Samples

Buccal Swab

 

 

Legend: Amplified Sigma products are resolved on a 1.5% agarose gel. 5 µl of amplified product was loaded per well. The GenomePlex amplified products result in an average size of 400 bp. The smear pattern varies by source as shown on the gel. Products amplified using GenomePlex technology are specific as evidenced by the lack of signal in the negative control (lane 3).

Lane 1—1 kb Marker
Lane 2—Positive Control
Lane 3—Negative Control
Lane 4—Corn

Lane 5—Ginseng
Lane 6—Purple Coneflower
Lane 7—Tomato
Lane 8—Soybean

 

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