Exercise 3 – DNA fingerprints of unknown DNA samples
You must work with a partner on this exercise. Step 1: Loading the gel. Obtain a sealed E-Gel from a lab instructor. Plug in the power base at your carrel. Open the package and insert the E-gel into the power base. The E-gel is inserted right edge first. Press firmly at the top and bottom until you hear it snap into place. A red light will steadily illuminate when the E-Gel cassette is properly inserted. If the red light does not come on, ask for help from a lab instructor.
Step 2: Uncovering the wells. The wells in the E-Gel are held open by a plastic comb. Remove the comb by carefully pulling it straight up. Dispose of the plastic comb in the refuse boxes provided in the Central Study Area. Do not reinsert the comb into the wells.
Step 3: Loading the wells. Obtain one “crime scene” and three “suspect” samples of DNA from a lab instructor. Using the micropipetting technique you practices in Exercise 2, dispense 20 ul of DNA from Suspect #1 into Well #2. Remember to hold the tip of the micropipette just inside the well. Take care not to puncture the bottom of the well.
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Using a fresh pipette tip for each sample, continue loading the remaining samples into wells # 4, 6 and 8, following the sequence below.
DNA sample from Sample vial Load into well number
Suspect #1 Clear 2
Suspect #2 Yellow 4
Suspect #3 Green 6
Suspect #4 Orange 8
Crime scene (Unknown)
Color __________ From Box # _____
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Step 4: Running the gel. After loading the E-Gel, push the button again – briefly – to start the 35-minute electrophoresis run. The light will change to a steady green light. Step 5: Turning off the unit. Current through the E-Gel will automatically cease at the end of 35 minutes. The E-Gel Power Base will signal the end of the run with a flashing red light and beeping. Press the button to turn the unit off. The beeping will continue for 5 minutes if it is not turned off. Step 6: Viewing the gel. Remove the E-Gel from the Power Base. Take it to the Central Study Area to view it under an ultraviolet (UV) light.
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Questions 1. Assume your gel results represent RFLP banding patterns of DNA fingerprints. Which DNA samples appear to have been derived from the same individual? Why? 2. What would the banding pattern of a DNA fingerprint look like if the investigator forgot to add restriction enzyme to the DNA samples before loading them into the gel? Show your TA (a) your test tube with extracted DNA from Exercise 1, and (b) your finished E-gel under UV light.