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Team:Macquarie Australia/Protocols/Agarose Electrophoresis
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Revision as of 04:42, 29 August 2013
Agarose Gel Electrophoresis
Preparing the gel:
Mix 10 g of agarose powder with 100 mL of 1 x TAE buffer and heat (requires to be qualified to use the microwave!). Add 1 μL of GelRed.
Pour mix into gel cast making sure of no leaks. Remove any bubbles with a pippette tip. NB. ensure cast is on a flat surface and balanced.
Place the appropiate comb into the cast (plan->how many wells will you need?). Leave gel to set. Place gel into the electrophoresis bath and fill with 1 x TAE buffer to cover.
Running the gel:
Generally a molecular marker will be loaded into the first and sometimes the last well(s). Given its high concentration take 1 μL of 1 kbp DNA ladder, 1 μL of 6 x dye (bromophenol blue) and 4 μL of 1 x TAE buffer (total = 6 μL).
Samples-> for most PCR products 5 μL can be mixed with 1 μL of dye (hence diluting the dye into a 1:6).
Tip->small sheet of parafilm can be used to mix the ladder/samples and the dye.
Load gel accordingly remembering to note what went into each well!
Run gel at 90V for 45 minutes using the dye as an indicator of the progress.
Photograph gels under UV light.
