Griffitts:Gel electrophoresis

This is the protocol for normal gel electrophoresis. The protocol for a low-melt gel for in-gel ligation is here.

Materials

 * Gel tray
 * 1X TAE buffer
 * 16-well comb
 * autoclave tape
 * agarose (for standard DNA gels)
 * 1 mg/mL ethidium bromide
 * 8X loading dye
 * DNA ladder

Gel preparation

 * Carefully tape both ends of the tray with autoclave tape
 * Insert the 16-well comb
 * Start with 50 mL 1X TAE buffer
 * Use the flask labeled "Agarose--keep in gel area"
 * Add 0.5 g agarose
 * Use the agarose labeled "for standard DNA gels"
 * This makes a 1% agarose gel; for other gels, adjust accordingly
 * Microwave until dissolved
 * This will take less than 1 minute and will require frequent stirring to prevent a boil-over
 * Add 8 μL of 1 mg/mL ethidium bromide and stir
 * This is kept in the fridge
 * Allow to cool ~5 minutes
 * Pour into the tray and allow agarose to solidify
 * Remove tape and comb
 * Remove the comb slowly so you don't tear the wells

Sample preparation

 * Cut a section of Parafilm
 * Add 2 μL 8X loading dye to the parafilm
 * Combine 2 μL of your sample to the 8X loading dye
 * Repeat this for each sample

Electrophoresis

 * Check to make sure there is enough 1X TAE buffer in the gel box
 * Dr. Griffitts has drawn a black line on the side indicating where is sufficient
 * If you suspect the 1X TAE buffer is old, replace it; you may dump the old 1X TAE buffer down the drain with water
 * Place your gel (still on the tray) in the box with the wells away from you
 * If your gel is pointing the wrong way, you will run your samples into the buffer
 * To the first lane at 5 μL of 1 kb DNA ladder
 * Add 4 μL of each of your samples (2 μL sample + 2 μL 8X loading dye) to each of the subsequent wells
 * Place the lid on the gel box
 * Make sure the black cable is away from you and the red cable is near you
 * If you reverse the cables you will run your samples into the buffer
 * Turn on the gel box
 * Adjust voltage to 120 V
 * Wait until you can see that the 8X loading dye has moved 1-2 inches down the gel
 * If you can't see your loading dye, you've probably run your samples into the buffer

Photography

 * Place your sample in the Gel Doc
 * You can leave it in the tray
 * Shut the door
 * Turn on the UV light
 * Adjust focus, zoom, and exposure time as needed
 * When you have the image how you want it, hit "Print" on the scanner
 * TURN OFF THE UV!
 * Remove your gel and throw it in the solid waste bucket
 * Wipe down the inside of the Gel Doc
 * Tear off your printed image