11.3: Gel Electrophoresis
Obtain simulated DNA samples and perform a gel electrophoresis generating a DNA fingerprint. Use this information to determine the guilt or innocence of the suspect.
Step 1: Setting up the agarose gel
- Obtain a gel former, comb, and masking tape. The instructor will demonstrate how to build the gel. Place the gel set-up in a safe location. Once the gel has been poured, do not move until solidified.
- Gently pour the agarose into the gel former until the level of the agarose is about 3⁄4 the length of the teeth of the comb. The tape will prevent the molten gel from spilling out of the apparatus. The agarose is kept in a 60 o C water bath to keep it molten. Agarose solidifies at room temperature so return the flask to the bath immediately after use.
Step 2: Preparation of samples and gel electrophoresis
- Obtain two simulated DNA samples A and B. The samples are not DNA but dyes that migrate in a manner similar to restriction fragments.
- Remove the tape from the gel former. Carefully remove the comb. Your instructor will demonstrate how to load the samples on the gel. Record which sample you load into which lane of the gel.
- Seal the top of the wells with a drop of molten agarose as demonstrated.
- Place the gel in the gel box. Samples are closest to the negative (black) electrode. Add 1X electrophoresis buffer to just cover the gel. Place the cover on the apparatus and connect the red and black leads (red is positive, black is negative).
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Notify your instructor at this point and s/he will turn on the voltage for you. The gel is run at ~90 volts for 20 minutes. Once the voltage is applied, observe that samples migrate towards the positive electrode (red). Do not leave the gel until you are sure that the samples are migrating in the correct direction.
* Complete Section10.5, Concept Review while the gel is running - Turn off the power supply. Unplug the leads. Visualize the bands of dye which represent the DNA fragments.
CLEAN UP:
Rinse gel equipment with water. Put gloves, paper towels in the regular trash. Dispose of electrophoresis buffer as instructed (you may recycle it, check with instructor). Wash hands.