Effects of Lysis Time and EDTA on Genomic DNA Prepared for Sequencing
Mentors: Kimberly Kanki & Marcia Lara
Genomic DNA (gDNA) that is isolated for DNA sequencing must be of high yield and high molecular weight. There are several factors that effect quality of gDNA, including length of lysis time, EDTA concentration, and tissue type/amount. Patricia’s goal was to determine whether the current standard protocol for isolating gDNA could be altered to improve DNA yield and molecular weight.
Patricia extracted gDNA from two different mouse tissues (liver and muscle) using three different lysis times and two different concentrations of EDTA. She then assayed DNA yield using PicoGreen, and DNA quality and molecular weight using both 2hr and pulsed field gel electrophoresis. Patricia’s results showed that there is no best combination of lysis time and [EDTA] that applies to all tissues. The current standard protocol results in the best yields, but with large amounts of variation between samples. Liver tissue always yields more gDNA than muscle tissue, and liver tissue is less sensitive to changes in lysis time and EDTA concentration than muscle tissue.
Patricia, a senior at Malden High School, determined conditions for extracting high yields of good quality DNA from different mouse tissues for DNA sequencing.