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Contents 1 DNA Gel Electrophoresis 1.1 Considerations: 1.2 Preparing the gel 1.3 Preparing the buffer 1.4 Preparing the gel electrophoresis trays

DNA Gel Electrophoresis

Considerations:

- Wear gloves at ALL times
- Keep ethidium bromide in one place and wear gloves when using it. Once you have finished using it OR are walking to other areas of lab, remove gloves and put on a fresh pair. ETHIDIUM BROMIDE IS A CARCINOGEN – DON’T SPREAD IT ALL OVER THE LAB!

Preparing the gel

• Make up a stock of agarose solution, in which the agarose should constitute 0.8% of the weight/volume (the rest should be made up of the buffer used for the DNA electrophoresis – see step 2 before proceeding)

• Melt the agarose solution. This should be done in a microwave, initially for 3 minutes on a medium/high setting. After this time has expired take out the solution using gloves and hold up to the light. Give it a swirl and check for any solids within the solution. If it hasn’t completely melted, put it back in the microwave until it does.

***Important note: when putting gel in microwave, keep checking on it regularly (once every 30-40 secs) – it must not be left unattended!***

• Using gloves, remove the gel from the microwave and check it for any solids. If it has completely melted, leave it somewhere to cool.

• Image 1 shows what the agarose solution should look like - as a clear liquid with no solid agarose present

Preparing the buffer

• The buffer we use for DNA gel electrophoresis is called TAE (Tris-acetate-EDTA) – WE NEVER USE WATER! To make up a stock solution of this buffer at 50x concentration TAE, use:

1. 242 grams of Tris
2. 57.1 ml of Glacial Acetic Acid
3. 100 ml of 0.5M EDTA (pH 8.0)

• Make up volume to 1 litre using DEIONISED WATER

• From this stock solution, make up a 1x concentrated TAE solution and to this add 8 microlitres of ethidium bromide.

Preparing the gel electrophoresis trays

• Make sure that a clean tray is prepared before carrying out procedure. This must be cleaned using de-ionised water and then with the buffer in which the electrophoresis will be conducted in.

• Make sure that the tray has been dried completely using a paper towel.

• Make sure that there is a clean, dry comb (cleaned in the same format as the tray).

• Insert the comb into the groove of the plate (nearest the end).

• Place the tray along with comb in proximity of the electrodes.

Image 4 shows the electrophoresis tray and the comb as separate items - make sure they are both cleaned and dried! Image 5 shows the manner in which the comb should be inserted into the electrophoresis tray - the groove near the edge of the tray should be used. This edge is where the DNA will be placed into the formed wells.