Automated Isolation of Genomic DNA from Large Volumes of Whole Blood
Part # PS165
Abstract
Sydnor Withers, Mary Dressler, Steve Krueger, Christine Helt, Eric Vincent and Cristopher Cowan
Promega Corporation, Madison, WI USA
A key source for genomic DNA (gDNA) is blood drawn into a standard 10ml Vacutainer® tube. The Promega ReliaPrep™ Large Volume HT gDNA Isolation System, integrated on Freedom EVO®- HSM workstation, provides a unique and dependable system for isolating genomic DNA from large volumes (1ml–10ml) of blood. The novel chemistry and instrumentation resolve many challenges encountered when processing large-volume samples in a high-throughput format such as: loss of sample pellets during decanting of fluids, transport of full 50ml tubes to various locations on a liquid handling robot, and manual re-suspension of final DNA pellets. Liquid handler resource constraints were removed by creation of a new accessory, the ReliaPrep™ HSM 32 LV instrument, which provides heating, shaking and magnetization of samples at one deck position. The combination of this device, the Freedom EVO®- HSM workstation and the ReliaPrep™ Large Volume HT gDNA Isolation System allows automated recovery of pure gDNA from up to 2 × 32 ten milliliter blood samples within 8 hours.
We present verification studies demonstrating automated system performance. Comparisons between the Freedom EVO®- HSM workstation and a standard precipitation-based method were made for duplicate blood samples. 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. Recovered DNA exhibited good purity with A260A280 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 many downstream analysis applications. Results of genomic DNA purification from frozen (hemolysed) blood samples and blood collected using common anticoagulants (EDTA, heparin, citrate) are also compared to demonstrate the efficacy of the new system.
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