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Whole Genome Amplification: Blood Card Protocol

Blood cards provide the convenience of archiving small volumes of blood. However, many times genomic DNA from these samples is limited, which may hinder the researcherā€™s ability to perform downstream analysis. This protocol provides a simple and convenient method to extract genomic DNA from a blood card. Once the DNA has been extracted, it can then be amplified using the GenomePlexĀ® Whole Genome Amplification protocol.

Required Products

  • GenEluteā„¢ Blood Genomic DNA Kit (Product No. NA2000)

Materials to be supplied by the User

  • Blood card
  • 1.5 ml microcentrifuge tubes
  • Ethanol (Product No. E7023)
  • Microcentrifuge (with rotor for 2 ml tubes)
  • Water, molecular biology reagent (Product No. W4502)
  • 55 Ā°C water bath or heat block

Extraction of DNA from a Blood Card

The GenElute Blood Genomic DNA Kit (Product No. NA2000) is recommended for this procedure.

  1. Cut a disc from a dried blood card (200 ĀµL spotted) into several 2 mm by 2 mm pieces and place the pieces into a 1.5 ml microcentrifuge tube.
  2. Add 40 ĀµL Proteinase K and 1.0 ml Resuspension Solution.
  3. Add 300 ĀµL of Lysis Solution C and vortex thoroughly for 15 seconds.
  4. Incubate at 55 Ā°C for 10 minutes.
  5. After the incubation, transfer the liquid (discard blood card remaining in the microcentrifuge tube) to a 15 ml conical tube.
  6. Add 500 ĀµL of Column Preparation Solution to the GenElute Miniprep Binding Column (red o-ring) and centrifuge at 12,000 Ɨ g for 1 minute.
  7. Discard the flow-through liquid.
    Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields.
  8. Add 900 ĀµL of 95ā€“100% ethanol to the lysate in the 15 ml conical tube and mix thoroughly by vortexing 5ā€“10 seconds.
  9. Transfer the contents of the tube into the treated column from step 6. Centrifuge at ā‰„6500 Ɨ g for 1 minute. Repeat until all of the lysate has been passed through the column.
  10. Discard the collection tube and flow-through. Place the column in a new 2 ml collection tube.
  11. Add 500 ĀµL of Prewash Solution (be sure to dilute with ethanol prior to first use) and centrifuge for 1 minute at ā‰„6,500 Ɨ g.
  12. Discard the collection tube containing the flow-through and place the binding column into a new 2 ml collection tube.
  13. Add 500 ĀµL of Wash Solution (be sure to dilute with ethanol prior to first use) to the binding column and centrifuge at maximum speed (12,000ā€“16,000 Ɨ g) for 3 minutes to dry the binding column.
  14. Pipette 400 ĀµL of Elution Solution onto the column and centrifuge for 1 minute at ā‰„6500 Ɨ g to elute the DNA.
  15. Store the eluted DNA at ā€“20 Ā°C or proceed to the amplification step.

Amplified Human Genomic DNA from Blood Card

blood_card

Legend: Products were amplified using the GenomePlex Whole Genome Amplification Kit (Product No. WGA1) from Sigma, Supplier Aā€™s kit and Supplier Qā€™s kit. Products were resolved on a 1.5% agarose gel. 5 ĀµL of amplified product was added to each well. The products amplified using GenomePlex technology were of a smaller molecular weight as shown on the gel when compared to Supplier A and Q. This is due to the random fragmentation of genomic DNA prior to amplification. Sigmaā€™s amplified products are specific and there is no amplicon visible in the negative control (lane 2) indicating that only the desired genomic DNA is amplified. Both Suppliers A and Q yield a nonspecific signal in the negative control which is equal in size and intensity to the signal for the suppliersā€™ positive control.

Lane 1ā€”1 kb Marker
Lane 2ā€”Sigma Positive Control
Lane 3ā€”Sigma Negative Control
Lane 4ā€”Sigma Blood Card
Lane 5ā€”Sigma Blood Card
Lane 6ā€”Supplier A Positive Control
Lane 7ā€”Supplier A Negative Control

Lane 8ā€”Supplier A Blood Card
Lane 9ā€”Supplier A Blood Card
Lane 10ā€”Supplier Q Positive Control
Lane 11ā€”Supplier Q Negative Control
Lane 12ā€”Supplier Q Blood Card
Lane 13ā€”Supplier Q Blood Card
Lane 14ā€”1 kb Marker

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