Agarose gel electrophoresis
Procedure
Preparation of Agarose Gel (1%)
1. Rinse and dry the gel casting tray with 95% ethanol. Place tape across each end of gel casting tray.
2. Insert well forming “comb” into slot on casting tray
3. Measure 1g of agarose and dissolve in 100mL of 1X TAE buffer by heating the suspension in a microwave oven until the agarose is completely dissolved and no lenses of agarose is seen floating in the solution
4. Cool the solution to around 50°C, add ethidium bromide (10 mg/mL) to a final concentration of 0.1 μg/mL or 1 μl per 10 mL of molten agarose.
Note:Do not pour the gel too hot as that may warp the gel former.
5. Pour in enough molten agarose so that teeth of comb are submerged and allow agarose to harden .This solidifying period should take at least 30 minutes.
6. Carefully remove comb and tape. Place the agarose gel with the tray into the electrophoresis chamber and add 1X TAE running buffer to cover the gel. Make sure the wells are submerged but do not over put buffer over the gel.
Electrophoresis of DNA samples
1. Add loading dye to sample. Dot 2 μl of 6X loading dye onto parafilm. Combine 10 μl of your DNA sample with the loading dye on the parafilm.
2. Carefully load 10-12 μL the sample into the preformed well of agarose gel and load 5 μL of DNA size standards (“1 kb DNA ladder”) into the far right or far left well.
3. Connect leads to power supply and the gels are run at 100 V for large gels and about 40 V for smaller gels (mini-gel).
4. Run until the tracking dye is about 2/3 of gel.
5. Shut off the power supply, disconnect the leads and remove gel in casting tray. Be careful that the gels can come off the trays easily and will break if they fall.
6.The gels can be viewed directly on a UV transilluminator (Gene Flash) if the DNA intercalating dye, ethidium bromide, has already been added, as in this case.
7. Otherwise, the gel is to be stained for 30 minutes in running buffer with 0.5 μg/mL ethidium bromide added before viewing on the transilluminator.
8. Photograph the gel.
