TITLE: 2% Agarose Gel Electrophoresis
AUTHORS: Alicia Grealy

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for electropohresing DNA through an agarose gel to examine fragment size. </div></div>

[STEPS]
?. [Cast a 2%, medium sized agarose gel]
Using an electronic balance, weigh out 2.2 g of agarose powder on to a weigh boat using a spatula.
Note that gel concentration can be adjusted. The more concentrated the gel, the greater the resolution of small fragment sizes.
?. [Cast a 2%, medium sized agarose gel]
Transfer the powder to a 200 ml conical flask.
?. [Cast a 2%, medium sized agarose gel]
Using the graduated measurng cylinder, measure out 110 ml of 1xTAE buffer. Add to the conical flask containing the agarose powder. Swirl the flask gently to mix.
?. [Cast a 2%, medium sized agarose gel]
Make up 2 L of 1X TAE buffer:            50 ml of 40X TAE buffer            1950 ml of MilliQ waterMix by inversion.
?. [Cast a 2%, medium sized agarose gel]
Microwave the flask uncovered for 1 minute.
A lid can loosly be placed over the flask but DO NOT tighten--allow steam to escape. Do not microwave for more than 1 minute at a time. Do not fill flasks or beakers/Schott bottles more than half full with liquid!
?. [Cast a 2%, medium sized agarose gel]
Remove the flask from the microwave using oven mitts and swirl gently.
The liquid is boiling! Use oven mitts to handle flask. Do not put your face over the opening to the flask as liquid can splash out!
?. [Cast a 2%, medium sized agarose gel]
Microwave the flask for a further minute but remove from the microwave if the agarose appears to boil excessively. Swirl to mix and examine near a light source to ensure the agarose has melted. Allow to cool for 5 minutes.
?. [Cast a 2%, medium sized agarose gel]
Place the gel casting tray into a rubber vice that will seal the ends tightly, or tape the ends with masking tape.
?. [Cast a 2%, medium sized agarose gel]
Place the assembly on a flat bench and use the spirit level to check it is level--adjust if needed.
?. [Cast a 2%, medium sized agarose gel]
Place a 20-well comb into the casting tray.
?. [Cast a 2%, medium sized agarose gel]
When the flask is cool to the touch, add 5 ul of SYBR Safe and swirl gently to mix. Avoid generating bubbles.
Wait until the liquid is warm (not boiling) to cast the gel, or the tray may crack!
?. [Cast a 2%, medium sized agarose gel]
Pour the liquid gel slowly into the casting tray. Pop any bubbles that have formed using a clean pipette tip.
?. [Cast a 2%, medium sized agarose gel]
Let the gel set for 20-30 minutes at room temperature.
?. [Cast a 2%, medium sized agarose gel]
Allow residual gel to set in the flask, then scrape into the bin. Fill the flask half full with water and microwave until the water boils. Pour the water down the sink and clean the flask using a bottle brush.
?. [Load and run the gel]
When the gel is set, remove the combs gently.
?. [Load and run the gel]
Place the casting try and gel in the electrophoresis tank.
?. [Load and run the gel]
Fill the electrophoresis tank with 1 X TAE buffer to the fill line indicated on the tank.
?. [Load and run the gel]
Pipette 3 ul of 50 bp DNA ladder into the first well of the gel.
The recommended volume will depend on the concentration of the ladder. Check the manufacterer's recommendations. If the ladder is not pre-mixed with loading dye, be sure to add 1-2 ul of loading dye before loading into the gel.
?. [Load and run the gel]
Place some Parafilm across a 96-well PCR plate rack and press down firmly to create small wells.
?. [Load and run the gel]
For each sample, pipette 1-2 ul of loading dye onto the Parafilm, taking care not to pierce the Parafilm.
?. [Load and run the gel]
Mix 10 ul of PCR product with the loading dye by pipetting gently up and down.
?. [Load and run the gel]
Transfer the 12 ul of PCR product/loading dye to the wells of the gel, taking care not to pierce the bottom of the well with the pipette tip.
The volume each well can take will depend on the size of the comb used. Be sure not to overload the wells or product will float out the top of the well.
?. [Load and run the gel]
Place the lid on the gel tank and plug the electrodes into the appropriate power slots. Ensure the positive electrode is at the base of the gel.
Take care when working with electricity and water! Check electrical cords of all equipment and ensure none are damaged and that cords are not a tripping hazard. Do not use if the electrical cord is damaged in any way. Tag the instrument with warnings, make the area safe, and notify your line manager and anyone else in the immediate area that may be affected. Use electrical equipment indoors only in an area free of explosive material, corrosive gas, powerful vibrations, direct exposure to sunlight, and temperature fluctuations. Use in a space where cables will not come into contact with liquids, be manually damaged, or interfere with other workplace operations. Do not use electrical equipment with any other power adapter or cord than the one supplied.
?. [Load and run the gel]
Switch the power pack on a set the voltage to 80 V and the time to 1 hr and 10 min.
Note that the voltage and time can be adjusted to suit what you are running on the gel. For amplicons (one small product), I will run the gel at 96 V for 30-40 min. For shotgun libraries, I will run the gel as above. The lower the voltage and longer it is run, the greater the separation of fragments will be.
?. [Load and run the gel]
Press 'Run' or 'Start' on the power pack and check to see that bubbles are rising from electrodes.
?. [Load and run the gel]
When the run is over, switch of the power pack, remove the lid, and remove the gel from the tank, taking care not to let it slide off the tray.
Do not remove the lid to the electrophoresis tank until the power pack is switched off.
?. [Photograph the gel]
Place the gel on the UV transilluminator and photograph using the attached camera. Follow the manufactuerer's instructions to use the equipment.
Take care working with UV. You should have UV safety training. Do not open the transilluminator while the UV is on! Use signage to warn others when the UV is on.
?. [Photograph the gel]
Discard the gel into a designated biohazard bin, and clean the UV dock with 70% ethanol.
?. [Finishing work]
Dispose of used tips into a designated sharps container.
?. [Finishing work]
Dispose of gel waste into a biohazard bag.
?. [Finishing work]
Used combs, beakers, flasks, and tray should be washed with warm water and placed on a rack to dry.
?. [Finishing work]
Gloves and chemical waste should be sealed in a biohazard bag for incineration.