Gel Electrophoresis

From FreeBio

{Note: The following protocol can be used for running DNA through an agarose gel.}

1. Make a 2% agarose gel. To a 250ml flask, add:
   • 3.0g agarose (high-melt, i.e. normal)
   • 150ml 1x TBE buffer
2. Microwave for ~1min and then for 10s blasts, to prevent boil over.
   {Note: To prevent the agarose from boiling over, do NOT secure the cap. To make clean up easier, place a piece of paper towel beneath the flask.}
3. Let stand for ~15min or until it stops steaming.
   {Note: Remember not to let it cool for too long!}
4. Add ~6ul ethidium bromide to molten agarose; swirl flask to mix.
   {Note: Ethidium bromide (EtBr) is a CARCINOGEN! Use caution.}
5. Set up mold box:
   • Make sure that the box is level.
   • Insert the gel mold. Tighten.
   • Insert the comb.
6. Pour gel and let stand to cool.
   {Note: The hardened gel will be opaque.}
7. Remove gel from mold and place in gel run box.
   {Note: Gels are slippery! Make sure it doesn't slip.}
8. Fill box with enough 1x TBE buffer to cover the gel.
9. Load the wells with desired DNA. In unused lanes place 10ul loading dye. For ladders, mix 1:10 ladder DNA, 1:10 loading dye, and 8:10 distilled H20.
10. Plug into power supply.
    {Note: Red to red, black to black! Remember that red means (+) and DNA runs toward (+). Make sure that's the end you want your DNA to be migrating towards.
11. Turn on power supply, set voltage to desired V.
    "{Note: 100V is good for a 150ml gel, 60V for a 60ml gel. It might do well to check the gel 5min after turning on the power supply to make sure that the DNA is running the right direction.}
12. Image when dye has run ~1/2 the length of the gel.