Pouring agarose gels is relatively straightforward, save for dealing with ethidium bromide. Wear gloves at all times, and dispose/exchange gloves liberally whenever in contact with an ethidium bromide containing surface. Dispose of all materials in marked containers and not in general waste containers. This page details how to make and pour an agarose gel, how to run a gel, and how to perform a gel extraction.

Pouring an Agarose Gel

This protocol is for a 1% agarose gel, which is suitable for sizes between 5 kbp to 500bp. Mass of agarose can be modified for differing percentage gels. Higher percentages are suitable for lower size products (i.e. 2kbp to 500bp) whereas lower percentages are suitable for larger products (i.e. 15kbp to 10 kbp).

Materials Required

  1. Ethidium Bromide (Sigma Aldrich E8751)
  2. Agarose (GoldBio A-201-100)
  3. Gel casting apparatus
  4. Gel running apparatus
  5. 1X TAE Buffer (Use Bio-Rad 1 L #1610743)
  6. Small, 100mL glass bottle with lid
  7. Small, 100ml glass beaker

Protocol

  1. Measure 0.5g of agarose using balance. Pour into glass bottle with lid. Modify mass up or down for a different percentage gel.
  2. Measure 50mL of 1X TAE Buffer and pour into glass bottle with lid. Turn cap until lightly closed, and then turn counter-clockwise one third turn to loosen so solution can vent.
  3. Microwave in series of 30s on high. Swirl each time vigorously between each stop. Stop when agarose is no longer forming molten balls and is dissolved. If solution boils over and out of bottle, start over.
  4. Wait until solution cools. Temperature should be hot enough that agarose is still dissolved and you can grasp with a gloved hand comfortably. If solid agarose appears re-heat until dissolved.
  5. While cooling, assemble gel casting apparatus and add combs.
  6. Pour into glass beaker. Add 5uL of ethidium bromide, swirl to mix and red ethidium bromide is totally dispersed.