Agarose Gel Electrophoresis
GOLD MEDAL WINNER 2008
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- Add 20ml 50 X TAE to 980ml H2O.
- Add 3g agarose powder to 300ml of the TAE-H2O (1 x TAE) solution.
- Microwave full power for 5 minutes or until solution is clear and agarose has dissolved.
- Leave to stand for 5-10 minutes. Put tape around sides of tray.
- Pour solution into tray, add 4μl of ethidium bromide and mix gently using the comb.
- Clean the comb (this prevents capillary action drawing the gel up between the comb teeth and making 'spikes' around the wells) and reinsert near one end of the tray.
- Leave to set 30-40 mins.
- Remove tape and comb, place tray in electrophoresis machine and pour 1 x TAE solution to just cover the gel.
- Combine 1μl of loading buffer for every 5μl sample being loaded and mix by pipetting up and down.
- Load marker and samples and run the gel.
N.B. We have found that a thin gel works best for loading small samples, but a thicker gel may be preferable if lots of sample needs to be loaded (for example if a fragment needs to be cut from the gel). A comb with teeth taped together using autoclave tape can also be used to create larger wells.
N.B. We have run gels at 100V for 20 minutes and 70V for 60 minutes. The latter setting/time is better for running larger fragments such as whole plasmids as a high voltage can cause shearing of the DNA.