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Higher gDNA Concentrations with the Perfect gDNA Blood Mini Kit ,,, by Varying the Final Elution Volume

Higher gDNA Concentrations with the Perfect gDNA Blood Mini Kit
by Varying the Final Elution Volume

Laura Pollock and Jennifer Halcomez
Eppendorf 5 Prime, Boulder, Colorado Introduction

The Eppendorf Perfect gDNA Blood Mini Kit protocol recommends an elution volume of 200 l for maximum total yield in standard application. In some instances, the concentration of the resulting gDNA may be more important than the total yield. Elution volumes of 50 l, 100 l, and 200 l were tested to determine the increase in concentration, as well as the potential effects on downstream applications. The kit was tested with blood from three different individuals using three different preservatives.

Materials

  • Perfect gDNA Blood Mini Kit
Required but not supplied:
  • Ethanol (95%100%); aerosol barrier pipette tips recommended; Microcentrifuge; Eppendorf Thermomixer, water bath, or heat block at 70C
Method

Isolation of gDNA:
  1. Pipette 20 l reconstituted Proteinase K into a 1.5 ml microcentrifuge tube. Add 200 l whole blood to the tube. Add 350 l Solution G1 to the tube containing Proteinase K and blood. Mix by vortexing for 5 seconds.
  2. Incubate in a Thermomixer at 70C, 900 rpm, for 10 minutes.
  3. Centrifuge the sample for 3 minutes at 12,000 to 16,000 x g (top speed in most microcentrifuges) to pellet cell debris. Pour the supernatant into a fresh microcentrifuge tube.
  4. Add 200
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