"
Team:Macquarie Australia/Protocols/Agarose Electrophoresis
From 2013.igem.org
(Created page with "{{Team:Macquarie_Australia/Style2}} {{Team:Macquarie_Australia/Header}} <center> <h1>Agarose Gel Electrophoresis</h1> <html> <b>Preparing the gel:</b> Mix 10 g of agarose pow...") |
|||
| Line 4: | Line 4: | ||
<center> | <center> | ||
<h1>Agarose Gel Electrophoresis</h1> | <h1>Agarose Gel Electrophoresis</h1> | ||
| + | </center> | ||
<html> | <html> | ||
| - | <b>Preparing the gel:</b> | + | <b>Preparing the gel:</b><br> |
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. | 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. | ||
| Line 13: | Line 14: | ||
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 (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. | + | 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.<br><br> |
| - | <b>Running the gel:</b> | + | <b>Running the gel:</b><br> |
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). | 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). | ||
Revision as of 04:41, 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.
