#49
DNA YIELDS FOR WB VS BUFFY COAT PROTOCOL UTILIZING THE GENOMTM-6.
Haecin Chun , Brett Peterson , Francesco Marincola MD , David Stroncek MD and Sharon Adams . 1 DTM-HLA Laboratory, NIH, Bethesda, MD, USA .
DNA isolation is a critical aspect of the modern day molecular lab. Not only must the isolation method produce pure high-quality DNA which is usable in all aspects of the laboratory; but, it is necessary for the isolation method to produce a sufficient quantity of DNA for all assays. Compared to other assays, PCR SSP HLA typing methodology requires some of the largest amounts of DNA. Since this is the method utilized by our laboratory, a goal was set to determine how to achieve the maximum yield from a GenoMTM-6.This robot has the ability to perform isolations on whole blood (WB) and buffy coat (BC) preparations. Standardization of BC prep was attempted due to manufacturer recommendations not to overload the instrument. In addition, WBC counts vary greatly depending on disease state of the patient. Therefore, comparisons were made between WB, WBC rich plasma preps, and standard BC preps. 350
L WB was tested using the standard WB protocol. 300L WBC rich and BC preps were tested using the BC protocol. All samples were <3 days old at the time of isolation. After extensive testing, WBC rich plasma preps were eliminated from comparison due to sample manipulation necessary to achieve an appropriate number of WBC. When manufacturer recommendations were followed to keep BC preps at WBC quantities 
5x106, DNA yields were comparable to WB preps. Further testing was done to test the limits of the BC protocol. Observations include that the reagents have a finite point where % yield falls and that the test system can handle WBC quantities >5 x106. Maximum yield attained from the system, thus far, is 26ug. The data collected provides information which each laboratory assessing the GenoMTM-6 can utilize to determine the most efficacious method for DNA isolation.