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PPP Lab Agarose Gel Protocol

  1. Determine the size gel you need to make, remember to account for at least one well for a ladder on each row:
    • Small gel comb capability: two combs with 14 wells each, total 28
    • Medium gel comb capability: two combs with 20 wells each, total 40
    • Large gel comb capability: two combs with 50 wells each, total 100
  2. For small gel box, make a 75mL gel mix, for the medium box make a 100mL mix.
  3. Making gels:
    For a 1% gel:
    • Small box: 75mL new 1X TAE buffer and .75g agarose
    • Med box: 100mL new 1X TAE buffer and 1g agarose
    • Large box: 150mL new 1X TAE buffer and 1.5g agarose
      For a 1.5% gel:
    • Small box: 75mL new 1X TAE buffer and 1.12g agarose
    • Med box: 100mL new 1X TAE buffer and 1.5g agarose
    • Large box: 150mL new 1X TAE buffer and 2.25g agarose
  4. Use a weigh boat designated for agarose and a scoopula to measure agarose and using the Erlenmeyer flask from the gel bench to measure the TAE
  5. Mix inside the Erlenmeyer flask: add the agarose by taco-ing the weigh boat and carefully pouring the powder as to not get it stuck to the side of the flask. Pour up to your desired volume with new 1X TAE. Swirl the flask to mix
  6. Put a scrunched Kimwipe in the mouth of the flask and put in the microwave for 1 minute. Every 20 seconds open microwave and swirl flask. Use an orange glove because glass gets hot
  7. Microwaving time can vary, so keep adding time until when you look at the liquid when swirling it is completely clear and there are no “flakes.” This is often when it is boiling, but you need to not let it continue boiling for too long because it will boil over out of the flask and into the microwave
  8. Add 5μl GelRed or 1μl GelGreen to the liquid and swirl the flask to mix thoroughly. GelGreen is preferred
  9. Set up gel cast mold, place your tray in the gel box sideways and make sure there is an air-tight seal with the rubber noodles. For the large gel box there is a separate casting mold. Use the level to make sure the tray is sitting level inside the box for a flat gel
  10. Let gel mix cool ~5 minutes in the flask before pouring into the gel tray, if you can safely hold it, that’s a good sign it’s ready
  11. Pour in the mix and put desired comb size into the tray
  12. Using a pipette tip, move any bubbles to the side of the tray
  13. Let harden until opaque and cool ~30min
  14. Take out and orient the gel with the comb side to the top of the box. Take the comb out of the hardened gel
  15. Pour enough “used” TAE buffer into the gel box to cover the gel with a thin layer of liquid and make sure there are no dimples where the wells are
  16. Add 3-4μl of the appropriate ladder to the first well in the gelOn a piece of parafilm, make dots of 1ul of loading dye for each one of your samples.
  17. On a piece of parafilm, make dots of 1ul of loading dye for each one of your samples. 1 by 1 for each of your sample aliquoits, add 5ul of sample DNA to the dot of loading dye and pipette to mix. It will stay in a bubble on the parafilm. Then suck up the full 6ul and add it to the gel
  18. Make sure you have made a map in your notebook that shows the order of samples
  19. Make sure gel box is set up to “run towards red”
  20. Plug black cable into black insert and red cable into red insert of the gel box
  21. Turn box on and make sure voltage is set to 80-100V. Set time for 45-60min
  22. Once done, set up the imager. Take the grey imager down from the shelf and remove the orange cover. Also take down the black imager shade and the small orange slide for imaging
  23. Slide your gel from the tray into the center of the blue square on the imager
  24. Place the black imager shade over the gel and the square and put the orange slide tile over the whole on the top
  25. Turn on the light and place your phone camera on the orange square so you can take a picture of your gel through the filter
  26. Once you are done with your gel, slide it off the imager and into the large black bucket in the cabinet under the gel bench. This is a non-hazardous waste container for only agarose and TAE
  27. Pour the leftover TAE from the gel box into the used TAE container using the funnel kept on the gel bench
  28. Rinse the gel box, comb, tray, and flask in DI type II water
  29. Wipe the imager with a Kimwipe and DI type II water
  30. Return all the materials you have used to where you found them
Written on March 1, 2019