DeRosa:Protocols/Agarose gel

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Supplies Agarose Low Melting [Fisher] TBE Deionized water Ethidium Bromide solution (Made from Fisher Biotech Ethidium Bromide) [Promega] 25 base pair ladder [Promega] Blue/Orange 6X Gel loading dye

Thermometer Erlenmeyer flask Microwave Agarose gel set tray & loading system: Owl Model A1 Gator Gel System (Large) OR i-Mupid mini gel 10uL pipetteman (Fisher or Gilson) & gel loading tips

AVOID The following -Taping the combs to be used as a wall of some sort, the sticky stuff stays stuck and ruins the comb -Allowing to gel to sit outside for long in warm weather -Do not prepare gels more than a few hours ahead of time (storing in fridge seems to produce poor results)

General Instructions: -For the mini gels use a 2.5% for the large gels look up in manual which percent to use for base pair size (generally used 2%) -Wear nitrile gloves as EtBr is toxic -Prepare a general 10mg/ml solution of Ethidium Bromide -Large Gel system instructions saved on V:\shared\DeRosaGroup\Manuals\Horiz Electro gel Owl.pdf -Small -iMupid gel system uses 50V, large Owl gel system uses 160V

Procedure:

  1. Weigh out desired amount of Agarose Low Melting (% of total gel volume)
  2. Add 1X TBE (Diluted accordingly with deionized water) and agarose to a large Erlenmeyer flask (at least 3 times the size of the volume of your solution). Plug the opening with a paper towel.
  3. Microwave for 1-3 minutes (depends on volume) until all agarose has dissolved without it boiling over
  4. Allow agarose solution to cool at room temperature until 60˚C (measure with a thermometer. Do not allow to cool much below 60˚C.)
  5. Add EtBr to a concentration of 0.5 ug/mL
  6. Slowly pour into gel tray (with the appropriate comb already inserted)
  7. Allow gel to solidify (Approximately 1 hour for a large gel)
  8. In the meanwhile, prepare loading samples in a small centrifuge or PCR tube and label

a. 1uL loading dye : 5uL DNA sample (or appropriate ladder)

  1. Once solidified, place gel with tray into system, remove combs and end guards and fill the system with TAE buffer to fill line. TBE buffer is preferred for large gels.
  2. Load 5uL of sample into the wells with gel loading barrier tips and a 10uL pipetteman
  3. Plug in appropriate power supply
  4. Run gel run
  5. Turn on Computer connected to Alpha Innotech gel visualizer
  6. Visualize on Alpha Nanotech UV gel visualizer, making sure the filter was on EtBr
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