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

Procedure

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