Polyacrylamide gel electrophoresis in PROTEAN TM-II (Bio-Rad) apparatus

Xiao-Ru Wang
  1. Turn on the cooling, it takes time to get the chamber cooled down
  2. Set up the gel plate
    * the glass plates should be clean with no grease spots on them
    * the "sandwich" should be fastened tight. A little space between the top edge of the outer glass plate and the holder should be kept to avoid the break down of the glass plates.
    * grease the two bottom corners to prevent leakage
  3. Prepare the polyacrylamide gel from stock solutions
    Gel concentration 8% 6%
    volume 100ml 50ml 100ml 50ml
    dH2O (ml) 52.7 26.4 59.3 29.7
    5X TBE (ml) 20 10 20 10
    30% acrylamide (ml) 26.6 13.3 20 10
    10% ammonium persulfate (ml) 0.7 0.35 0.7
    0.35 TEMED (ul) 35 ul 17.5 ul 35 ul 17.5 ul
    * mix H2O, 5X TBE buffer and 30% acrylamide according to the proportion listed above
    * de-gas the mixture
    * add 10% ammonium persulfate and TEMED, mix gently
    * slowly fill the polyacrylamide solution into the gel plate with a 50ml syringe
    * insert the comb, be careful not to trap air bubbles underneath the comb teeth
    * polymerization takes about 40 - 60 min; a heating lamp will shorten this step
    * remove the comb from the gel and immediately add water into the slots; wash the slots with distilled water 2 - 3 times; fill the slots with 1 X TBE buffer, the gel is ready for loading samples.
  4. Load the DNA samples into the gel
    * add 1.2 ul of loading buffer III into 10 ul of DNA samples
    * apply the samples with a syringe
  5. Electrophoresis
    * fix the gel plate on the cooling unit
    * pure 1.5 L of 1X TBE buffer into the bottom chamber
    * transfer the gel/cooling plates into the chamber; avoid air bubbles on the bottom of the gel plate
    * fill in the top chamber with 1X TBE buffer
    * connect the apparatus to the power supply
    * run-in the sample at 20 - 25 mA
    * run the gel at 5 - 6 W (voltage x current) till the front bromophenol blue reaches the end of the gel
    Stock solutions
    30% acrylamide acrylamide
    29 g bis-acrylamide 1 g
    H2O to 100 ml
    * filter, store the solution in dark bottle at 4 C or room temperature, up to one month
    * unpolymerized acrylamide solution is toxic, handle with care
    10% ammonium persulfate: ammonium persulfate 1 g make to 10 ml with dH2O (solution can be stored at 4 C for several weeks)
    Silver staining of DNA in the polyacrylamide gel
    Fixation
    2x 7 min in: 10% EtOH 100ml + acetic acid 0.5ml
    Wash 3 times with dH2O
    Silver impregnation 40 min in 100ml of 0.15% AgNO3
  6. Wash 3 times with dH2O
  7. Image development
    NaOH 1.5 g
    0.756% Na(BH4) 1 ml
    formaldehyde 1 ml
    H2O to 100 ml.
    This step takes about 5 - 10 min and can be controlled by eye.
  8. Wash the gel 2x with dH2O
  9. Keep the gel in gel-fixer for one day and then dry up
    Gel-fixer
    30 ml glycerol; 200 ml EtOH; 270 ml H2O
    Stock solutions
    10% EtOH 0.15% AgNO3: 0.15g AgNO3 in 100 ml H2O.
    Store in dark bottle.
    0.756% Na(BH4): 0.076g NaBH4 in 10 ml of H2O

by Alfred E. Szmidt