Published: 21-07-2020| Version 1 | DOI: 10.17632/shvj3728p6.1
Alexandre Fonseca


Software to acquire RGB-based electropherograms using a web-cam. Applied for quantitative determination of coloured compounds. Instructions to use the software RGB-GE 1- After installation, run RGB Gel Electrophoresis package from the Windows Start Menu. 2- Connect a webcam to the PC and press the “CAMERA ON” button to choose the appropriate device to perform the analysis. 3- Press the “CAMERA SETTINGS” button to set camera properties according to the apparatus you are using. We recommend setting a proper image contrast between gel (as white as possible) and separated coloured spots. 4- Adjust the size and position of pixel delimiters (9 rectangles on the image acquired by the webcam in real time) using the numeric up and down buttons “Length”, “Width”, “X coordinate”, “Y Coordinate” and “Y Gap”. All delimiters change position and size simultaneously. Be sure that delimiters are bigger than the larger spot and placed them at the right of the first spots to be detect. This is necessary because delimiters will move from right to left during the measurements. 5- Set “MAX x” and “MAX y” at plot settings. 6- Set the number of readings and the number of replicates to be considered during the acquisition of the electropherograms. The number of readings refers to the displacement (in pixels) from the right side to the left side of the image, necessary to perform signal acquisition for all separated spots. The number of replicates is used to set the amount of images to be considered for the signal average. In other words, if 10 is selected, the average for R, G and B signals detected for 10 sequential images of the same delimiter at the same position will be considered as signal for analytical purposes. 7- Press “Start” button to initiate the acquisition of the electropherograms. Select a folder and give a name to the file. During measurements, data is automatically saved in .txt format with the first column representing pixels displacement (X coordinate) and the following columns representing the responses for R, G and B for the first delimiter, then R, G and B for the second delimiter and so on until. 8- Press “Shot” button to perform the readings of the spots. When the number of readings is reached, press “shot” again for a new acquisition. If readings are performed during the electrophoretic run, press shot until the appropriate electropherograms are collected. 9- Open the .txt file with a suitable package as Origin or Excel to estimate peak areas.