Team:CUNY Kingsborough/Experiments
Experiments
Ethidium Bromide Spot Test
Accurate and quick determination of DNA concentration is critical for the assembly of synthetic constructs, as well as a multitude of other experiments. We sought to optimize an under-utilized and inexpensive approach for determining DNA concentration: a spotting technique that uses the intercalating dye Ethidium Bromide. This technique does not require specialized equipment such as a spectrophotometer, but instead relies on visualization of dye-DNA complex fluorescence when excited by UV light. We modelled and tested a range of parameters for dye concentration and spot size, finding that 15uL spots with 1.0ug/mL Ethidium Bromide produced the most reliable standard curve. A detailed paper with our findings from 2017 data can be found here: Quantification of DNA samples by Ethidium Bromide Spot Technique.
Protocol
Below is the procedure used for data collection in 2017 and what we sent to iGEM teams in 2018 as part of Collaboration:
5-15uL of diluted Ethidium Bromide was pipetted onto a disposable plastic petri dish. 1uL of water or diluted lambda DNA was then pipetted into the droplet/spot containing the dye and mixed. The dish was then inverted over a UV transilluminator. A cover shielded ambient light and a photograph was taken using an orange light filter and an android smartphone (Motorola Droid 2).
Image Processing
Images of the spots were processed using ImageJ®. Briefly, spots were selected using a freehand or oval tool, either to analyze the entire spot(Whole Area) or the region within the spot with the brightest uniform intensity (Highest Fluorescence). Pixel intensity was measured using the analysis tool, with or without a de-speckling correction applied first (Noise reduction). These measurements were either taken as a ratio to a background signal and/or normalized to the maximum value for a set, and averaged across multiple experiments.