Team:BCCS-Bristol/Protocols-Agarose Gel Electrophoresis

Agarose gel electrophoresis

Gel preparation:

- For a thick gel in a small chamber, boil 50 ml 0.5x TBE in the microwave (for a thinner gel to insert only 5 µl sample in each well 40 ml are sufficient)

- Cool the solution until you can touch it with your hands for a longer time

- Add 0.5 µl ethidium bromide per 10 ml TBE and mix while avoiding air bubbles

- Pour the gel into a chamber with a comb that you prepared before

- Let the gel cool down and become solid (15-20 min)

Sample preparation:

- Add 10 % “Sample Loading Buffer” (BIORAD) to the sample

- Mix gently and spin shortly down

Loading the gel:

- Remove the comb and put the gel with the slide into a chamber (the wells need to be on the end with the black pole!!!)

- Fill the chamber with 0.5x TBE until the gel is covered

- Use 5 µl HyperLadderI (BIOLINE)

- For colony PCR, put 5 µl of each reaction in the well

- Close the chamber with the lid (black pole to black=negatively charged and red to red=positively charged…)

- Run the gel with 80-100 V

The Sample Loading Buffer contains two dyes: Bromophenol blue runs at ~300 bp and Xylene cyanol FF runs at ~4 kb.