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PAGEgelCasting.md

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Denaturing PAGE with BioRad Mini-PROTEAN setup

For casting 2 gels.

Casting the Gel

components from Bio-RAD

  1. Mini-PROTEAN gelbox, vertical tank, lid, and power-supply
  2. 0.75mm, 12-well combs
  3. Glass backplate for 0.75mm gels
  4. Glass front plate
  5. Casting mount to hold gel plates together
  6. Casting stand to seal bottom of gel plates while they set
  7. Casting stand

Reagents (25 mL of gel 15% Denaturing Gel)

  • 6g Urea
  • 3.5 mL 40% acrylamide (19:1) Bis
  • 2.5 mL 5x TBE
  • 1.5 mL ddH2O
  • 40 uL 10% APS (ammonium-persulfate). Make fresh from powder
  • 20 uL TEMED

Procedure

  • weigh out urea in 100 uL beaker
  • add acrylamide, TBE and ddH2O
  • add stir bar,
  • stir and heat on low gently 3-5 minutes, until Urea is dissolved. Let cool.
  • Add 20 uL of TEMED and 20-40 uL of 10% APS. Mix thoroughly!
  • Pour gels. Makes 2 0.75 mm gels.
  • Note: Polymerization will happen in a few minutes to an 1 hour after adding APS.
    • If it is too fast to allow you to pour the gels, use less APS next time.
    • Allow gels to fully polymerize before removing from casting unit. Use the excess gel as a test for when polymerization is complete. If not polymerized in a hour, repeat and use 2x APS.
  • Unused gels may be stored for a few days to a week wrapped in Seran wrap with a moist paper towel backing.
    • These can last longer if kept cold, however sometimes 4C causes the Urea to crash out of solution, so RT may be better.
    • Old gels may start to frown.

Running Gel

  • Warm 1 L of 1x TBE buffer for 2:30 min in microwave.
  • Turn on water-bath to 60C. Fill water bath with enough water to rise to the same level of buffer in the BioRad tank.
  • Insert gel into gel box and load box into tank. Pre-run gel for 10-30 minutes at 120 V to equilibrate gel temperature.
  • Denature samples in loading buffer (e.g. 2x Urea-loading buffer from BioRad) at 90C for 2-5 min. Return to water-ice bath.
  • Flush extra urea from wells prior to loading.
  • Load sample, run gel for 50 minutes at 120 V.
  • Gently extract gel from casing. Incubate in 1x Gel-Green in 1x TBE for 10-30 minutes.
  • Image Gel on Typhoon Imager.

Notes:

Comb volumes

  • 10-well (30 µl),
  • 15-well (15 µl),
  • prep well (450 µl),
  • 10-well (50 µl),
  • 12-well (20 µl),
  • 9-well (30 µl),
  • IPG comb (7 cm IPG strip)

Gel volumes

  • 0.75 mm 4.2 mL
  • 1.0 mm 5.6 mL
  • 1.5 mm 8.4 mL