CONCENTRATED DNA PREPARATIONS FROM COMMERCIAL ISOLATION KITS: MODIFICATION OF ELUTION VOLUME AND BUFFER
R Cirocco, M Markou, C Gomez, W Smith, V Esquinazi, A Tzakis and J Miller
Department of Surgery, Division of Transplantation, University of Miami School of Medicine and the Veterans Affairs Administration Hospital Miami, Fla.

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Most commercial DNA isolation kits use a set volume of their proprietary elution buffer to elute or reconstitute the final DNA prep from whole blood. Three major disadvantages in these procedures are; 1) the final DNA concentration may be too low for Sequence Specific Priming or other HLA PCR amplification procedures, 2) the elution buffer may not be suitable for long term storage and 3) The patients’ white Blood Cell (WBC) count may be too low and the set elution volume will dilute the DNA to a sub optimal concentration. We have modified the Qiagen Blood Isolation kit (Valencia, CA. ) by using 25% of the buffer recommended for elution. In this procedure the manufactures recommends using 200 micro liters of their elution buffer and then to repeat this elution procedure. This results in genomic DNA preps with a concentration of 34 ng/ul for one elution and 17 ng /ul for the two elutions of 200ul. By using 50ul of sterile K buffer (1X PCR buffer,0.05M KCl, 0.025M MgCl, 0.01 M Tris Ph 8.0, containing 0.5% tween twenty) we have obtained elutions in the range of 80-117 ng/ul of genomic DNA. An ideal concentration for commercial SSP kits is 80 to 120 ng/ul. In addition the elution buffer is at a pH that maintains the DNA structure and reduces hydrolysis that can occur in acidic conditions. An ASHI requirement is that a lab stores DNA at conditions that retain the fidelity of the DNA. We have tested these preps (N=14) for up to three years and they still yield excellent PCR amplification. The resulting high concentrations of DNA and use of this elution buffer, fulfill those requirements. An unknown buffer solution may not. In conclusion we have modified an existing procedure from a commercial kit for DNA isolation in a way that results in a genomic DNA preparation that has a high concentration of DNA, is in a buffer suitable for long-term storage, and ideal for PCR amplification.