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Materials

• Agarose
• 1× TAE (Tris-acetate–EDTA buffer)
  • 50× TAE: 40 mM Tris base, 20 mM acetic acid, 2 mM EDTA, pH 8.5
  • Dilute 50× TAE to 1× for use. Add 80 mL empty carboy and fill to 4 L with diH₂O.
• Bottle or flask
  
  
• Gel caster
• Gel tray of desired length
• Gel comb of desired size/thickness
• Electrophoresis chamber with clean-enough 1× TAE
• Electrophoresis power supply
• Microwave oven
• Gel viewing equipment (blue-light source and orange glasses, or gel imager)

Procedure

Gel Preparation

1. Weigh and mix agarose powder with 1× TAE to desired

1. agarose concentration in a flask or bottle. An flask for agarose is usually by the gel station. Make enough volume for however many gels at their respective concentrations.
   Alternatively, obtain previously prepared solid agarose gel in bottle/flask.
   • Agarose gels are commonly used in concentrations of 0.7–2%m/V, depending on the expected size of bands needing to be resolved. See table.
2. With a loosened cap, microwave agarose suspension or solid until completely clear and

1. homogeneous liquid (≈1 min per 100 mL on high). Watch as it boils to stop the microwave

1. before it can overflow. Swirl, handling with a silicone mit. Continue microwaving in

1. smaller increments if necessary.
   If

1. needed, dilute molten agarose with 1× TAE to desired percentage, swirling to mix. Make enough for the desired casts and the level you wish to fill. See below.

1. • E.g. you can keep a bottle of 2% agarose and melt it

1. • when needed, diluting with TAE in a second flask to the desired 0.

1. • 7–2% concentration. Pouring in a second container and dilution also cools it, so the poured gel solidifies faster.
2. If

1. prestaining the gel, to a final 1× add 10,000× DNA dye (GelGreen or GelRed) to the molten agarose and swirl until color is uniform

1. .
   • Dye can be incorporated into the gel during preparation (prestaining), after gel electrophoresis by incubating in a dye bath (poststaining), or solely added to the DNA sample (sample dying). Prestaining is most robust. Post-staining requires more time and monitoring. Sample staining impacts DNA migration, so is not best for analytical purposes and must be used with a similarly dyed ladder.)
2. Position tray(s) in center of clamp and tighten clamp moderately. Place desired comb(s) in tray slots, oriented so the prongs are closer to the top of the gel. Ensure tray is level using bubble level, adjusting gel caster's screw feet.
   • Pick gel casting trays appropriate for your use. The short tray holds 40 mL when filled to the top of comb

1. • prongs. The long tray holds 55 mL when filled to the top of the comb

1. • prongs.
     For gel purification purposes, you may want a thick gel (

1. • tray filled to top of comb prongs) to minimize the number of wells needed to hold the samples. For analytical purposes, you may need only a thin

1. • gel (casted to half prong height) to hold small volumes of samples such as analytical DNA digests.
   • Pick comb appropriate for your use. Thinner combs are superior for analytical purposes, as they result in thin bands more accurately assessable. Thicker combs are superior for purification purposes, as they hold more sample. A full-height narrow well holds ≈30 µL; a full-height wide well holds ≈60 µL.
2. Pour molten agarose in

1. trays to desired level while still hot, but not boiling to prevent tray/clamp warping. Use comb to pull bubbles and impurities away to

1. end of gel.
2. Allow the gel to solidify at room temperature or level in the deli fridge

1. 8–15 min.
   • Pre-chilled caster/clamp speeds solidification.

Sample Preparation, Loading, and Electrophoresis

1. Mix samples with 6× loading dye (loading buffer). If sample-staining, ensure the 6× loading dye contains 1× GelGreen/GelRed.
   • Loading dye does not typically contain any DNA dye, only tracking dyes that allow estimation DNA migration progression without fluorescence. Loading dye also contains glycerol or PEG to weigh down the sample in the well.
2. Remove comb from cast gel and submerge gel with tray in an electrophoresis chamber, oriented with wells at the top and the negative (black) terminals of the chamber at the top.
   If TAE doesn't cover the gel, add sufficient TAE. 
   • Gels can be cut to save lanes for later use. The tray isn't always necessary. Gels must be stored in TAE + DNA dye (if prestained), TAE (if unstained), or wrapped ≈airtight in plastic wrap.
   • If chamber liquid is cloudy or has crystals, empty contents into gel liquid waste tank. Gently rinse chamber in sink without pointing stream at deep ends, where it can damage the filament. Do a final rinse with diH2O. Refill with 1×TAE.
3. Load samples and, finally, ladder into wells. Pre-dyed ladder is generally at the gel station.
   • Pipetting into the bottom of the well allows more sample to fit in a well.
4. Secure the lid onto the chamber. Plug in lid into power supply, matching terminal colors. Ensure negative, black terminals are at the top of the chamber and lid and red at the bottom.
   Program in voltage (80–150 V) and time (15–45 min) and run. Confirm running by spotting bubbles coming from the terminals.
   
   • 120 V for ≈25 min is sufficient for most applications. Higher voltage shortens run time, but can sometimes result in band smearing or other problems due to heating.
   • The lower, blue/purple band migrates as ≈300 bp DNA, and should typically migrate ≈75% of the lane length before ending.
5. When the run is over, if poststaining, stain the gel in TAE + 1× DNA dye solution, rocking for 20–30 min, followed by destaining in water if necessary.
   Inspect/cut the gel under blue light with orange glasses or UV with UV shield. Image the gel with the blue light (or UV) imager: Open Canon EOS utility on the computer to awaken the connected camera and use Remote Live View to adjust the gel position and autofocus before taking a picture. The camera should typically be in the No Flash setting (auto-set exposure, ISO, and aperture). Exit EOS Utility when done.
   • Blue light is safer to work with and less damaging to DNA than UV light.