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Experiment 6: Agarose Gel Electrophoresis

1. Preparing an Agarose Gel 1. Prepare the running buffer stock solution: a. 10x TBE Buffer is prepared using 108g Tris base, 55g Boric acid and 9.3 g Na2EDTA b. The solution is topped up to 1L with DI water, pH should be 8.3 c. 10x TBE Buffer is diluted to make 1x TBE Buffer 2. Preparation of a 1% agarose gel: a. 0.6g agarose is dissolved in 60 mL of 1x TBE buffer b. Heat to boiling on a hotplate c. Add 2 ul of Gel Green stain and let cool to 55OC stirring constantly d. Keep cool in water bath 3. Ensure rubber gaskets fit snugly against the sides of the rig to prevent leaks 4. Insert the comb into the slots on the end of the tray and ensure that it is completely vertical 5. Get a flask of molten 1% agarose gel with Gel Green stain from the 55OC water bath and pour the entire contents into the tray 6. Allow the gel to solidify for 20 minutes 7. Once the gel is set, obtain approximately 450 mL of 1x TBE from the carboy and pour a small amount onto the surface of the gel 8. Slowly and gently pull the comb up and straight out of the gel 9. Lift the tray out of the rig and rotate 90O so that the wells are at the top, with the electrodes on the right side of the rig. The cathode should be at the top end, by the walls, and the anode should be at the bottom end of the rig 10. Fill both sides of the tank with 1x TBE buffer until the gel is covered by 2 mm of buffer

2. Loading an Agarose Gel 1. Obtain one tube of the following: a. Genomic DNA b. Restriction enzyme treated genomic DNA c. Plasmid DNA d. Restriction enzyme treated plasmid DNA e. λHindIII ladder 2. using the P20 pipette set to 20 ul, load the DNA samples and one of the two ladders. Use a lane lay-out sheet to record which sample is loaded into which lane 3. Once all the lanes have been loaded, slide the lid onto the tank until the power supply leads are securely attached to the banana plug electrodes. Ensure that the cables are inserted in the correct jacks on the power pack. 4. Do not allow your loaded gel to sit too long before beginning electrophoresis

3. Running an Agarose Gel 1. Turn on the power pack and set the voltage to 115-120V. Ensure that bubbles form at both ends of the tank 2. Let the gel run for 1 hour or until the dye font has moved 5 cm from the wells 3. Turn the power off and slide the lid off the tank. Pick up the gel on the tray, holding the open ends so the gel does not slide off the tray. 4. Drain as much buffer off the tray as possible. Gently slide the gel into the weight boat 5. After viewing the gel on the blue light box, drain the buffer into the waste container and rinse the tank, tray and comb.

4. Viewing an Agarose Gel 1. Take gel to the blue light box 2. Discard the gel into appropriate discard container 3. Rinse the weight boat and leave it to air dry...