Team:Calgary/Sandbox/Notebook/Protocols/SDSPAGEGel

From 2013.igem.org

SDS Page Gel

Reagents and Materials

  • SDS PAGE Gel apparatus: lid, tank, combos, spacer plates, short plates, casting frame and casting stand

15% SDS PAGE Gel (Separating)

  • 1.725 mL H2O
  • 1.875 mL Acryl/Bis (40%)
  • 1.3 mL 1.5M Tris (pH 8.8)
  • 50 µL 10% SDS
  • 50 µL 10% APS
  • 2 µL TEMED

10% SDS PAGE Gel (Separating - for large proteins)

  • 2.35 mL H2O
  • 1.25 mL Acryl/Bis (40%)
  • 1.3 mL 1.5M Tris (pH 8.8)
  • 50 µL 10% SDS
  • 50 µL 10% APS
  • 2 µL TEMED

7.5% SDS PAGE Gel (Separating - for our largest constructs)

  • 2.66 mL H2O
  • 0.938 mL Acryl/Bis (40%)
  • 1.3 mL 1.5M Tris (pH 8.8)
  • 50 µL 10% SDS
  • 50 µL 10% APS
  • 2 µL TEMED

12% SDS PAGE Gel (Separating - for Western Blot)

  • 5.0 mL H2O
  • 5.0 mL Acryl/Bis (30%)
  • 4.0 mL 0.5M Tris (pH 6.8)
  • 150 µL 10% SDS
  • 75 µL 10% APS
  • 7.5 µL TEMED

5% SDS PAGE Gel (Stacking)

  • 1.395 mL H2O
  • 0.375 mL Acryl/Bis (40%)
  • 1.17 mL 0.5M Tris (pH 6.8)
  • 30 µL 10% SDS
  • 30 µL 10% APS
  • 3 µL TEMED

4% SDS PAGE Gel (Stacking - for Western Blot)

  • 9.0 mL H2O
  • 2.0 mL Acryl/Bis (30%)
  • 4.0 mL 0.5M Tris (pH 6.8)
  • 150 µL 10% SDS
  • 150 µL 10% APS
  • 15 µL TEMED

Protocol

  1. Put two gel glass pieces together in the correct orientation (should be small space between them) and clip into the gel apparatus
  2. Add about 5 mL of separating gel in between the two glass pieces (ensure it does not leak)
  3. Add a thin layer of isopropanol on top of the separating gel to form a smooth line
  4. Wait for the gel to solidify (~ 20 minutes)
  5. Rinse out the isopropanol with water
  6. Add about 2 mL of the stacking gel on top of the separating gel and add the gel comb
  7. Wait for the gel to solidify (~ 20 minutes)
  8. Place the gel into the gel running apparatus and lock it in place. Ensure the side with the larger piece of glass is facing outward. If there is only one gel, add empty glass pieces to the other side
  9. Put the gel into the gel box and fill with 1x SDS Running Buffer. Ensure the middle section between the gels is filled with buffer
  10. Load samples
  11. Run gel at 100V until samples leave the well and increase the voltage to 150V. Or run the gel at lower voltages (~90V) to get cleaner bands
  12. When the gel is done running, fix it with destaining solution (50% water, 40% methanol, 10% acetic acid) for 30 min
  13. Stain the gel with Coomassie Brilliant Blue for 1h
  14. Distain the gel with water or destaining solution

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