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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