Components

Preparation

1. Thoroughly rinse gel housing and well-comb with dH₂O.

2. Place gel mold perpendicular to flow direction, ensuring proper sealing of rubber gaskets.

CRITICALIf the gaskets are not sealed properly, the molten agarose will flow into the gel housing and be very difficult to clean/remove.

3. Add the calculated amounts of 0.5xTBE and agarose to a fresh Erlenmeyer flask.

Do not discard the foil from autoclaving the flask, it can be used to recover the flask.

4. Heat in microwave until mixture can be dissolved.

CRITICALDo not let the mixture boil over and out of the flask.

Typical heating time for 50mL in a 2.45GHz microwave oven at full power is 30s.

5. Replace foil over mouth of flask, gently swirl until well mixed.

6. Allow gel to cool, gently swirling periodically until ~55°C.

7. Add appropriate ethidium bromide volume, swirl until uniform.

CRITICALCheck gloves for punctures or carefully double-glove before handling ethidium bromide!

CRITICALImmediately dispose of pipette tip directly to biohazard waste.

8. Gently pour molten agarose gel into housing, avoiding air bubbles.

9. Place desired well comb in desired position.

10. Once gelled, carefully remove well comb in a uniform fashion.

11. Remove gel mold and replace in parallel direction to flow.

12. Fill each reservoir with 0.5xTBE, continue to fill until gel is covered ~0.5 cm from 0.5xTBE surface.

13. Once the gel is no longer needed, it should be disposed of directly to the biohazard disposal.

CRITICALCheck gloves for punctures or carefully double-glove before handling ethidium bromide-containing gel!