Maheshri:Gel

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Casting an agarose gel

  1. Make 1x TAE buffer by diluting the 50X stock. You will need ~350mL for the small Bio-Rad gel box and ~1000mL for the medium Bio-Rad gel box.
  2. Add agarose and 1x TAE buffer to a 250mL Erlenmeyer flask. Add sufficient agarose to 30-40mL of TAE for small gels or 80-100mL of TAE for medium gels. A 0.8% agarose gel is usually good for most purposes since it can resolve DNA fragments of 0.5-10kb. Cast a 1.5% or 2.0% gel if you need to resolve smaller fragments (0.1-0.5kb).
  3. Microwave TAE and agarose until the gel starts to bubble and becomes transparent. Be careful not to overboil. Take out the flask and let it cool down until you can touch the flask with bare hands. Add Ethidium bromide (EtBr) and swirl the flask to mix. The EtBr stock is 20,000X so add 1μL per 20mL of agarose gel solution. Dispose the EtBr contaminated tip in the designated waste container located in the chemical fume hood.
  4. Place the gel tray in the casting device. The two ends of the tray should be tightly sealed. Place combs of appropriate width and number into the tray.
  5. Pour the gel mixture into the tray. Rinse out the flask with water immediately after pouring.
  6. Let the gel sit for at least 15 minutes. After the gel has solidified, remove the combs, lift the gel tray and put it into the gel box. Add 1X TAE to the chamber until the buffer just covers the gel.

Running electrophoresis

  1. Load 5-10μL of DNA ladder. To make 1mL of 1kb plus ladder, mix
    • 20μL 1Kb Plus DNA Ladder from Invitrogen (@ 1 μg/μl)
    • 100μL 10X Orange-G DNA loading dye
    • 880μL ddH2O
  2. Add 10X Orange-G loading dye to the DNA samples (e.g. use 2μL of dye per 20μL of sample). Mix with pipetting.
  3. Load samples carefully (and slowly) into the wells.
  4. Cover the gel box with the lid such that the negatively charged DNA samples will run towards the positive, red electrode. Make sure that the cables are properly connected to the power supply.
  5. Turn on the power supply and run your gel at 110V for 30 mins. If you are in a hurry, run the gel at a higher voltage (but not exceeding 130V). You should see bubbles rising from the electrodes if the gel is running properly.

Visualizing gels

  1. Lift the gel tray and allow the residual buffer to drip back into the gel box.
  2. Place the gel tray in the center of the UVP Biodoc-it imaging system. Click the "Live" button such that the gel displays on screen. Adjust the focus/zoom of the camera so that the entire gel is within the frame.
  3. Close the door and turn on the transilluminating UV light.
  4. Click the "Capture" button to capture the image appearing on screen. You may need to increase/decrease exposure time to improve image. Once the image is captured, turn off the UV light.
  5. Press the "Save" button to save the image. Make sure the SD card is inserted into the imager.
  6. To excise gel pieces, use a pipette tip to hold the safety trigger for the imager's door in order to keep the UV light on while having the door open. Wear a face shield or UV protective goggles.
  7. After imaging, remove gel and wipe down UV box if necessary.
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