Whole Genome Amplification

Animal Tissue

 

Animal tissue is a common source of material when performing genetic analysis. The protocol below is a simple method of extracting DNA from the animal sample. Once the DNA has been isolated, it can then be amplified using the GenomePlex® Whole Genome Amplification Protocol. The GenomePlex products have been used to amplify genomic DNA from chicken, porcine, bovine, fish, and shrimp sources.

Note: The protocol below describes the process of DNA purification and subsequent whole genome amplification. We have dramatically streamlined this process with our GenomePlex Tissue Whole Genome Amplification Kit (Product No. WGA5). This kit includes optimized reagents for tissue disruption and cell lysis eliminating the need for DNA purification prior to amplification. For more information on WGA5 Amplification Protocol see Whole Genome Amplification Directly from Archived Tissue.

Required Products

  • GenElute™ Mammalian Genomic DNA Miniprep Kit (Product No. G1N10)

 

Materials to be Supplied by the User

  • Animal tissue
  • Microcentrifuge (with rotor for 2 ml tubes)
  • Ethanol (Product No. E7023)
  • 55 °C water bath or heat block
  • 70 °C water bath or heat block
  • Water, molecular biology reagent (Product No. W4502)

 

Extraction of DNA from Animal Tissue
It is suggested to use the GenElute Mammalian Genomic DNA Miniprep Kit (Product No. G1N10) for this procedure.

  1. Place 20 mg of tissue into a microcentrifuge tube.
  2. Digest Tissue: Resuspend the tissue pellet in 180 µl of Lysis Solution T.
  3. Add 20 µl of proteinase K, mix by vortexing.
  4. Incubate at 55 °C for 2–4 hours or overnight until the tissue is completely lysed. Vortex occasionally during incubation.
    Optional RNase treatment: If residual RNA is a concern, add 20 µl of RNase A solution and incubate at room temperature for 2 minutes.
  5. Lyse cells: Vortex for 15 seconds. Add 200 µl of Lysis Solution C to the sample. Vortex thoroughly as a homogenous mixture is essential for efficient lysis. Incubate at 70 °C for 10 minutes.
  6. Prepare column: Add 500 µl of the Column Preparation Solution to each pre-assembled GenElute MiniPrep Binding Column and centrifuge at 12,000 × g for 1 minute.
  7. Prepare for binding: Add 200 µl of ethanol to the lysed sample and mix by vortexing.
  8. Load lysate: Transfer the entire contents of the sample tube into the treated binding column from step 5. Centrifuge at ≥6500 × g for 1 minute. Discard the collection tube containing the flow-through liquid and place the binding column in a new 2 ml collection tube.
  9. First wash: Prior to first use, dilute the Wash Solution Concentrate with ethanol as described under preparation instructions. Add 500 µl of Wash Solution to the binding column and centrifuge for 1 minute at >6500 × g. Discard the collection tube containing flow-through liquid and place the binding column in a new 2 ml collection tube.
  10. Second wash: Add another 500 µl of Wash Solution to the binding column and centrifuge for 3 minutes at maximum speed (12,000–18,000 × g) to dry the binding column. It is crucial that the binding column is free of ethanol before eluting DNA off the column. Centrifuge the column for an additional minute if residual ethanol is visible. Finally, discard the collection tube containing the flow-through liquid and place the binding column in a new 2 ml collection tube.
  11. Elute DNA: Pipette 200 ml of the Elution Solution directly into the center of the binding column and incubate at room temperature for 5 minutes. Centrifuge for 1 minute at ≥6500 × g to elute the DNA.
  12. Store DNA samples at –20 °C.

 

Application Data

GenomePlex Whole Genome Amplification Performed on Various Animal Tissues

FFPE

 

 

Legend: Amplified Sigma products are visualized on 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 animal 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—Porcine



Lane 5—Bovine
Lane 6—Chicken
Lane 7—Catfish
Lane 8—Shrimp



 

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