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Animal Tissue DNA-Extraction & WGA Amplification Protocol

Introduction

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 Whole Genome Amplification method using WGA1 and WGA2 kits. The GenomePlex products have been used to amplify genomic DNA from chicken, porcine, bovine, fish, and shrimp sources.

It is suggested to use the GenElute Mammalian Genomic DNA Miniprep Kit (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 μl 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.

WGA Amplification

Protocol for GenomePlex Whole Genome Amplification performed with GenomePlex Whole Genome Amplification Kit (WGA1) and/or Complete Whole Genome Amplification Kit (WGA2).

Fragmentation

  1. Prepare DNA solution of 1 ng/ml from whole blood extraction protocol described above.
  2. Add 1 µl of 10X Fragmentation Buffer to 10 µl DNA (1 ng/µl) in a PCR tube.
  3. Place the tube in a thermal cycler at 95 °C for exactly 4 minutes.
    Note: the incubation is time sensitive and any deviation may alter results. 
  4. Immediately cool the sample on ice and centrifuge briefly.

Library Preparation

  1. Add 2 µl of 1x Library Preparation Buffer. 
  2. Add 1 µl of Library Stabilization Solution.
  3. Mix thoroughly and place in thermal cycler at 95 °C for 2 minutes.
  4. Cool the sample on ice and centrifuge briefly.
  5. Add 1 µl Library Preparation Enzyme, mix thoroughly, and centrifuge briefly.
  6. Place sample in thermal cycler and incubate as follows:
       16 °C for 20 minutes
       24 °C for 20 minutes
       37 °C for 20 minutes
       75 °C for 5 minutes
       4 °C hold
  7. Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at - 20 °C up to three days.

Amplification

  1. Add the following reagents to the entire 15 ml reaction:
       7.5 µl 10x Amplification Master Mix
       47.5 µl Nuclease Free Water
       5.0 µl JumpStart Taq DNA Polymerase (12.5 units) for WGA1
       -or-
       5.0 µl WGA DNA Polymerase for WGA2
  2. Mix thoroughly, centrifuge briefly, and begin thermocycling:
       Initial Denaturation:  95 °C for 3 minutes
       Perform 14 cycles as follows:   
       Denature:  95 °C for 15 seconds
       Anneal/Extend:  65 °C for 5 minutes
  3. After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification.

Reamplification

  1. Add 10 µl of 1 ng/µl WGA amplified DNA to a PCR tube or multiwell plate.
    Note: It is necessary to clean up the WGA reaction to decrease possible bias in the reamplification. We recommend using the Sigma's GenElute™ PCR Clean-Up Kit (Product Number NA1020) or standard purification methods that isolate single and double stranded DNA.
  2. Create amplification mix. For each reamplification reaction, add the following to the WGA amplified DNA (step 1):
        47.5 µl of Nuclease-Free Water
        7.5 µl of 10X Amplification Master Mix
        5 µl of WGA DNA Polymerase
  3. Vortex thoroughly, centrifuge briefly, and begin thermocycling. The following profile has been optimized for a PE 9700 or equivalent thermocycler:
        Initial Denaturation 95 °C for 3 minutes
        Perform 14 cycles as follows:
        Denature 94 °C for 15 seconds
        Anneal/Extend 65 °C for 5 minutes
  4. After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification. The stability of WGA DNA is equivalent to genomic DNA stored under the same conditions.

Purification

Purification of Amplified Products performed with GenElute PCR Clean-Up Kit (NA1020)

  1. Insert a GenElute Miniprep Binding Column (with a blue O-ring) into a provided collection tube, if not already assembled. Add 0.5 ml of the Column Preparation Solution to each miniprep column and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the eluate.
    Note: The Column Preparation Solution maximizes binding of the DNA to the membrane resulting in more consistent yields.
  2. Add 5 volumes of Binding Solution to 1 volume of the PCR reaction and mix. For example, add 500 µl of Binding Solution to 100 µl of the PCR reaction. Transfer the solution into the binding column. Centrifuge the column at maximum speed (12,000 to 16,000 x g) for 1 minute. Discard the eluate, but retain the collection tube.
  3. Replace the binding column into the collection tube. Apply 0.5 ml of diluted Wash Solution to the column and centrifuge at maximum speed for 1 minute. Discard the eluate, but retain the collection tube.
    Note: Be sure to add ethanol to the Wash Solution Concentrate prior to first time use. See Preparation Instructions.
  4. Replace the column into the collection tube. Centrifuge the column at maximum speed for 2 minutes, without any additional wash solution, to remove excess ethanol. Discard any residual eluate as well as the collection tube.
  5. Transfer the column to a fresh 2 ml collection tube. Apply 50 µl of Elution Solution or water to the center of each column. Incubate at room temperature for 1 minute.
    Note: When eluting with water, make sure that the pH of the water is between 5.5 and 8.5. Elution may also be performed using the Elution Solution diluted 10-fold with water.
  6. To elute the DNA, centrifuge the column at maximum speed for 1 minute. The PCR amplification product is now present in the eluate and is ready for immediate use or storage at –20 °C.

Quantification of Amplified Products

The amount of DNA amplified using Whole Genome Amplification can be detected with or without purification. For the highest quality samples of DNA, it is strongly recommended to purify the samples after amplification. The amplified products can be measured with the PicoGreen® dsDNA Quantitation Assay from Molecular Probes Inc. (#P-7589). Another method of detecting the amplified products is spectrophotometric absorption (OD260) on a NanoDrop® spectrophotometer. This instrument can measure absorbance on 1 μl of sample over a large dynamic range, from 2–3700 ng/

Application Data

Whole Genome Amplification Performed on Various Animal Tissues

Amplified Sigma products are visualized on 1.5% agarose gel. 5 μl of amplified product was loaded per well. The WGA amplified products result in an average size of 400 bp. The smear pattern varies by animal as shown on the gel. Products amplified using WGA 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

Materials

     

Materials to be Supplied by the User

  • Animal tissue sample
  • 1.5 ml microcentrifuge tubes
  • Microcentrifuge (with rotor for 2 ml tubes)
  • 55 °C water bath or heat block

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