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Team:Macquarie Australia/Protocols/Agarose Electrophoresis
From 2013.igem.org
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<h1>Agarose Gel Electrophoresis</h1> | <h1>Agarose Gel Electrophoresis</h1> | ||
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<b><font size = 2>Preparing the gel:</font></b><br> | <b><font size = 2>Preparing the gel:</font></b><br> | ||
| - | Mix 1 g of agarose powder with 100 mL of 1 x TAE buffer and heat | + | Mix 1 g of agarose powder with 100 mL of 1 x TAE buffer and heat for 1 minute. Swirl and continue to heat in 10 second cycles swirling in between until solution is dissolved. 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. | 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 | + | Place the appropiate comb into the cast. Leave gel to set. Place gel into the electrophoresis bath and fill with 1 x TAE buffer to cover.<br><br> |
| + | <center><img src = https://static.igem.org/mediawiki/2013/7/7a/Photo%281%29.JPG></center> | ||
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<b><font size = 2>Running the gel:</font></b><br> | <b><font size = 2>Running the gel:</font></b><br> | ||
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Run gel at 90V for 45 minutes using the dye as an indicator of the progress. | Run gel at 90V for 45 minutes using the dye as an indicator of the progress. | ||
| - | Photograph gels under UV light. | + | Photograph gels under UV light.<br><br> |
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| + | <center><img src = https://static.igem.org/mediawiki/2013/8/8a/Photo%282%29.JPG></center> | ||
Latest revision as of 01:54, 26 September 2013
Agarose Gel Electrophoresis
Preparing the gel:
Mix 1 g of agarose powder with 100 mL of 1 x TAE buffer and heat for 1 minute. Swirl and continue to heat in 10 second cycles swirling in between until solution is dissolved. 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. 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.
