Agarose Gel Electrophoresis

What you will need:

• 1X TAE
• Graduated cylinder
• 250 mL flask
• Agarose
• Gel forming tray
• Ethidium bromide

Procedure:

• Dilute stock of 50X TAE to 1X with ddH2O.
• Measure 70 mL of buffer for small gels (large gels 170 ml).
• Transfer buffer to 250 mL flask (or 500 ml).
• Weigh out enough agarose to make 1% gel. (1% of 70 mL is 0.70 g)
• Transfer agarose to flask. Form an improvised cap by inverting 50 ml flask into neck.
• Melt agarose in microwave, stirring ever 15-20 seconds. This should take about 2 min.
• Allow agarose to cool.
• While agarose is cooling, assemble gel pouring apparatus by inserting gate into slots. Use a pasteur pipet to run a bead of molten agarose along the inner and outer edges of the gates to help seal the box and prevent leaks.
• Allow gel to cool until flask can be handled comfortably.
• Place comb in the gel rig.
• Pour agarose into gel tray.
• Allow to solidify completely. While the gel is solidifying, prepare the samples. Add your sample and 2uL of 10x OG loading dye to a tube, then make the total volume of the tube up to 20 uL. Or 2.2 ul of 10x OG to 20 ul sample.
• Pour 1X TAE over gel so that gel is covered by 3-5 mm of buffer.
• Load samples into lane. Do not forget to load 1kb+ ladder into one of the lanes.
• Hook electrodes to gel apparatus. Nucleic acids are negatively charged, so they will run to the positive (red) terminal.
• Pipette 10 uL ethidium bromide (from 10 mg/ml stock) into the buffer at the bottom of the gel. Mix well
• Turn on the gel. Run for 60 min @ 90V. Check with handheld UV Source.
• Place gel in plastic wrap.
• Carry to G311.
• With bare hands log in as Gen 420 with password Molecular1.
• Double click on the Genesnap from Syngene icon.
• Click on the Green Button to start live image.
• Put one glove on your left hand and place gel on transilluminator. Now do not touch anything with your left hand.
• With your right hand slide the door down completely.
• The transilluminator image on the screen should turn purple.
• Use the arrows on the exposure button to increase the exposure time until the gel and bands are clearly visible.
• If necessary use the zoom arrows to increase or decrease the size of the gel.
• Reposition the gel if necessary – open the door with your right hand and move the gel with your gloved left hand.
• To fine focus the image use the eye arrows.
• When the image is sized and focused properly capture it by clicking the red button.
• Print a photograph by clicking the printer icon in the tool bar at the top.
• Record your use on the sheet. Supervisor=iGEM and your initials.
• Log off.
• Remove your gel and clean the transilluminator with water and dry with paper towels.
• Take the gel back to the lab and dispose of properly.

Notes

• Ethidium bromide is carcinogenic!