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Agarose gel electrophoresis

Overview

Agarose gel forms when a solution containing disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose cools down below 35C and non-covalent bonds allow a porous structure to form (100-300 nm size pores). Best used with DNA fragments >100 bp.

Source: Extraction, Modification and Biomedical Application of Agarose Hydrogels: A Review (https://www.mdpi.com/1660-3397/21/5/299) (Source: Jiang et al., 2023)

Gel preparation

  1. Choose gel percentage based on the DNA size that you want to resolve and measure X g of agarose to make X% (w/v) agarose gel (usually 1 or 2%):
Gel % DNA size range (bp)
.5 1000 - 30000
.8 800 - 12000
1.0 500 - 10000
1.5 200 - 3000
2.0 50 - 2000
  1. Mix agarose powder into 100 ml 1X TAE or TBE buffer in a microwavable flask (while shaking the flask) and let it hydrate for a minute or two.
    • TBE: Best for smaller DNA fragments (<1500 bp), used with a higher voltage, can run for a longer time due to a higher buffering capacity
    • TAE: Best for larger DNA fragments (>4000 bp), used with lower voltage, good for purification from gel.
  2. Microwave for ~2 min with the bottle cap only losely holding until the agarose is completely dissolved.
    • Do it in pulses: 30-45 sec microwave, stop and swirl (with a heat-resistant glove on), repeat until no more crystals are visible.
  3. Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins.
    • Place the bottle briefly under running tap water to speed up cooling, but don't overdo it!
  4. (Optional) Add 5 uL of SYBR Safe to prestain.
    • Only for linear fragments because this dye acts as an intercalator and might alter plasmid migration, but should not affect linear fragments.
  5. Pour the agarose into a gel tray with the well comb in place.
    • Make sure there are no bubbles – use a pipette tip to move them to the sides. Bubbles are most likely to appear near the comb.
  6. Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.
    • Cover with a piece of foil to avoid dust.

Running

  1. Put the gel tray into the gel tank.
  2. Fill gel box with 1X TBE (or TAE) until the gel is covered (important to use the same buffer).
  3. Add samples:
  4. 6 uL of sample with loading buffer: 1 uL TriTrack + 5 uL DNA (mix and spin down)
  5. 3 uL ladder (mix gently)
  6. Run at 100V for 1% agarose gel (40 min) or at 120V for 2% agarose (30 min)
    • Rule of thumb: 5-10 V/cm of gel. Small size gel is 7 x 10 cm, so 35-70 V, but people just use 100V.
    • It should start bubbling immediately.
    • In a few minutes, you should see some bands
    • At the end, the yellow die should be at the bottom of the well
  7. Proceed to staining.

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