Introduction

A protocol on how to prepare and run a standard agarose gel electrophoresis at a certain percentage of agarose. The percentage of agarose to use can be estimated according to the sequence length (bp)1.

Materials

• 10X TAE buffer
• SYBR Safe (10 000X)
• Distilled water
• DNA samples (PCR,..)
• Parafilm
• Gel Loading Dye, Purple (6X) (NEB)
• GeneRuler 1 kb Plus DNA Ladder (ladder should be adapted to the sequence length)

Procedure

Preparation of the gel

1. Prepare 60 ml of (1-2)% Agarose in 1X TAE buffer.
• Dilute the 10X concentrated TAE buffer by adding 6 ml to 54 ml of water in a column.
• Weight the desired amount of agarose (1% := 600 mg) and put it in an Erlenmeyer. Add the buffer.
• Melt the agarose in a microwave oven (no aluminium or Parafilm) for around 2 minutes (until all the agarose is dissolved).
2. Add 6 µl SYBR safe into the Erlenmeyer.
3. Set up the gel-casting mold into the frame (rubber ends against the walls). Place the combs onto the frame at the top of the mold.
4. Pour the agarose solution into the gel-cast (don't overfill) and wait until the gel solidifies before loading your samples (between 1/2 and 1 hour).
5. IMPORTANT: Place the gel-cast so that the holes are directed towards the negative terminal (black wire).
6. Cover the gel with 1X TAE buffer (~250 ml) and CAREFULLY remove the combs.

Sample loading

7. On a parafilm, mix 8 μl of your DNA samples with the amount of loading dye needed for a total reaction volume of 12 µl, then load your samples and 5 µl of DNA ladder separately on different wells.
8. Run the gel at 100 Volts for 30-40 minutes.
9. Remove the gel from the chamber and take a photography at the UV transilluminator.

References

Promega-What percentage agarose is needed to sufficiently resolve my DNA sample?