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Hello everyone! Welcome to the Immunology lab at SASTRA. Let us start with today's experiment. Weigh 100mg of agar and dissolve it in 10 ml of 1x Running Buffer. Melt the Agar to Dissolve it properly using a Microwave. Add Antibody to the molten agar. Add it to clean labelled glass plate. Add agar slowly without bubbles. punch holes. Sample Preparation Label four microtest tubes: 1:2, 1:4, 1:8, and 1:16. Using a micropipette, add 50 microliters of Buffer to each tube. With a fresh pipet tip, add 50 microliters of “Standard” to the tube labeled 1:2, mix. Transfer 50 microliters of the 1:2 dilution to the tube labeled 1:4, Mix, and so on. Procedure Deliver specimen or standard (5 μl) to well by placing the pipette tip at the bottom of the well. Allow the well to fill to the top of the agar surface. Avoid bubbles to ensure proper volume and diffusion of sample. Visualization may be aided by placing the plate on dark background. Mark time of completion on plate cover and replace cover. Replace plate in bag and reseal carefully. Incubate plates upright on a flat surface at room temperature (20 to 24 oC) over 48 hours for end-point readings. Calibration Using the reference sera determine their ring diameters to the nearest 0.1mm. Using regular graph paper plot the concentration on the X axis and the zone diameters squared on the Y axis for each protein for End point readings. Draw a straight line of "best fit" between the four points. Results Measure diameters of precipitin zones to within 0.1mm. Variations in incubation times of more than 30 minutes will produce changes in diameters, especially those at higher levels of antigen except when plates have reached endpoint. Determine the concentration of each unknown or specimen protein from the reference curve find the corresponding concentration. Thank you and have a nice day!