Whole Genome Amplification

WGA1 & WGA2 Amplification

Whole genome amplification is performed with GenomePlex Whole Genome Amplification Kit (Product No. WGA1) and/or GenomePlex Complete Whole Genome Amplification Kit (Product No. WGA2).

Random Fragmentation

  1. Prepare DNA solution of 1 ng/µl from extraction protocol described above.
  2. Add 1 µl of 10× Fragmentation Buffer to 10 µl DNA (1 ng/µl) in a PCR tube.
  3. Place the tube in a thermal cycler at 94 °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.

 

OmniPlex Library Preparation

  1. Add 2 µl of 1× 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 ml of 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.
  8. Samples may be amplified immediately or stored at –20 °C for up to three days.

 

Whole Genome Amplification

  1. Add the following reagents to the entire 15 µl reaction:
    7.5 µl 10× 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 94 °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.

 

 

Achieve Robust Amplification Representative of the Original Input Genome

Amplification Image

 

GenomePlex WGA was performed on genomic DNA isolated from HT29 colon carcinoma cells and from a healthy human male. 2.5 µg of WGA product was labeled with Cy3 or Cy5 dye using the Genomic DNA Labeling Kit PLUS (Agilent). The entire labeled sample was loaded onto an Agilent Human Genome CGH Microarray 105A. Specific activities were between 28 and 43 for all samples, and always within 50% of samples being compared. The dye swaps (A & B) demonstrate that there was no bias in the DNA labeling and the aberrations detected are consistent with the HT-29 karyotype.

 

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