I previously checked Claire’s sheared DNA on the Bioanalyzer to verify the fragment size and to quantify the samples. Looking at her notebook, her numbers differ greatly from the Bioanalyzer, possibly due to the fact that the DNA1000 assay chip used only measures DNA fragments up to 1000bp in size. If her shearing was incomplete, then there would be DNA fragments larger than 1000bp that wouldn’t have been measured by the Bioanalyzer. So, I decided to quantify the samples on the NanoDrop1000 (ThermoFisher) again.
Results:
Spreadsheet: 20150226_Claire_sheared_Emma_1000ppm_OD260s
(http://eagle.fish.washington.edu/Arabidopsis/20150226_Claire_sheared_Emma_1000ppm_plots.JPG)
Comparison of NanoDrop1000 and Bioanalyzer measurements.
| Sample | NanoDrop (ng/μL) | Bioanalyzer (ng/μL) |
| 2M sheared | 48.03 | 16.28 |
| 4M sheared | 190.96 | 58.52 |
| 6M sheared | 141.56 | 42.98 |
| 2MHS sheared | 221.93 | 32.45 |
| 4MHS sheared | 257.48 | 43.82 |
| 6MHS sheared | 202.02 | 51.12 |
The NanoDrop is known to overestimate sample quantities due to the indiscriminate nature of UV spectrophotometry and that could be the reason for the large discrepancy between the two measurements (i.e. RNA carryover may lead to overestimation). As such, I’ll quantify the samples using a fluorescence-based assay for double stranded DNA tomorrow in hopes of getting the most accurate measurement.