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Protocol for GenomePlex Whole Genome Amplification ,,,from Whole Blood

  • Add the following reagents to the entire 15 l reaction:
    • 7.5 l 10x Amplification Master Mix
    • 47.5 l Nuclease Free Water
    • 5.0 l JumpStart Taq DNA Polymerase (12.5 units)
  • 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
  • After cycling is complete, maintain the reactions at 4 C or store at -20 C until ready for analysis or purification. VI. Quantification of Amplified Products
    The amount of GenomePlex Whole Genome Amplification Kit products can be detected with or without purification. For the highest quality samples of DNA we strongly recommend cleaning up the samples after amplification. The amplified products can be measured with the PicoGreen dsDNA Quantitation Assay (Molecular Probes Inc. Product # P-7589). Another method of detecting the amplified products is spectrophotometric absorption (OD260) on a NanoDrop instrument.

    VII. 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 l of Binding Solution to 100 l of the PCR reaction. Transfer the solution into the binding column. Centrifuge the column at maximum speed (12,000-
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