Analysis of DNA fragments using the Agilent 2100 Bioanalyzer
During the development of DNA shearing protocols for Covaris Focused-ultrasonicators, the Agilent DNA 12000 kit was used to evaluate the average base pair (peak) size because of the large fragment size range this kit covers, encompassing the 130 bp to 5 kb range achievable with a Covaris system. In the protocols developed by Covaris, the target DNA fragment base pair size is determined using the Agilent DNA 12000 kit to identify the maximum amplitude (Peak) of the electropherogram. When performing DNA analysis using the Agilent High Sensitivity DNA kit, the shape of the curves are quite different in comparison to the DNA 12000 kit results, particularly with DNA fragments 400 base pairs (bp) and larger. This result could be expected, since the two kits employ different gel matrices, causing the same size DNA fragments to migrate differently. When using the High Sensitivity DNA kit, the maximum amplitude method can lead to an overestimation of the average fragment size. When assessing the size of sheared DNA using the Agilent High Sensitivity DNA kit, a smear analysis is the more effective method to determine the average fragment size.