Griffitts:Gel electrophoresis

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

Contents

Materials

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
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