Whole Genome Amplification

Buccal Swab


This protocol provides a simple and convenient method to isolate, amplify, and purify genomic DNA from buccal swabs. Buccal swabs are a convenient method of acquiring a DNA sample. Once the DNA is isolated using the following extraction protocol, it can be amplified using the GenomePlex® Whole Genome Amplification Protocol to provide enough DNA for numerous downstream applications.

Required Products

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

 

Materials to be Supplied by the User

  • Buccal swab
  • 1.5 ml microcentrifuge tubes
  • 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 Buccal Swab
It is recommended to use the GenElute Mammalian Genomic DNA Miniprep Kit (Product No. G1N10) for this process.

  1. Dry the collected swabs at room temperature for 15 minutes.
  2. Add 280 µl of Lysis Solution T and 20 µl of Proteinase K. Insert the swab and gently spin. Cap the tube and mix by vortexing.
  3. Incubate the sample at 55 °C for 20 minutes with occasional vortexing.
  4. Add 200 µl of Lysis Solution C and vortex thoroughly for 15 seconds.
  5. Incubate at 70 °C for 10 minutes.
  6. Add 500 µl of Column Preparation Solution to each GenElute Miniprep Binding Column (red
    O-ring) and centrifuge at 12,000 × g for 1 minute. Discard the flow-though liquid.
    Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields.
  7. Add 200 µl of 95–100% ethanol to the lysate from step 5.
  8. Mix thoroughly by vortexing and add the entire contents of the tube into the binding column.
  9. Centrifuge at ≥6500 × g for 1 minute.
  10. Discard the collection tube containing the flow-through and place the binding column in a new 2 ml collection tube.
  11. Add 500 µl of Wash Solution (be sure to dilute with ethanol prior to first use) and centrifuge at
    ≥6500 × g for 1 minute.
  12. Discard the collection tube and flow-through and place the binding column in a new 2 ml collection tube.
  13. Add another 500 µl of Wash Solution to the binding column and centrifuge at maximum speed (12,000–16,000 × g) for 3 minutes to dry the binding column.
  14. Pipette 200 µl of Elution Solution onto the binding column and centrifuge for 1 minute at
    ≥6500 × g.
  15. Store the eluted DNA at –20 °C or proceed to the amplification step.

 

Application Data

GenomePlex Whole Genome Amplification Performed on a Buccal Swab Sample

Buccal Swab

 

 

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 source as shown on the gel.

Lane 1—1 kb Ladder
Lane 2—Blood
Lane 3—Plant
Lane 4—Buccal Swab

Lane 5—Soil
Lane 6—Positive Control
Lane 7—1 kb Ladder

 

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