Sydnor Withers, Ph.D., Mary Dressler, Steve Krueger, Christine Helt, Eric Vincent, Ph.D. and Cristopher Cowan, Ph.D.
Promega Corporation, Madison, WI USA.
The Promega ReliaPrep™ Large Volume HT gDNA Isolation System, integrated on the Tecan Freedom EVO®- HSM workstation, provides a unique and dependable system for isolating genomic DNA from blood fractions derived from 2.5ml - 10ml of whole blood. The combination of Promega’s ReliaPrep HSM 32 LV instrument, which provides heating, shaking and magnetization of samples at one deck position, the Freedom EVO®- HSM workstation and the ReliaPrep Large Volume HT gDNA Isolation System allows automated recovery of pure gDNA from 32 blood fraction samples in any combination of sample volumes allowed for each sample type within 4 hours.
Here we present verification studies demonstrating automated system performance from whole blood, buffy coats, red cell pellets, and all cell pellets. Comparisons between the Freedom EVO®- HSM workstation and a standard precipitation-based method were made for quadruplicate blood samples from multiple donors. Yield, purity, and integrity of extracted gDNA were assessed using UV absorbance spectroscopy and gel electrophoresis. Genomic DNA yields from normal 10ml whole blood samples were 200 to 400μg (depending on white blood cell count) in an eluted volume of 1ml. DNA yields for all cell pellets and buffy coats were similar to the corresponding whole blood yields while red cell pellet yields reflected the effect of the white blood cell removal. Recovered DNA exhibited good purity with A260/A280 ratios greater than 1.7 and A260/A230 ratios between 1.8 and 2.2. Isolated DNA was suitable for storage and was used in multiple downstream applications.