Acrylamide gels
Pouring Tris-Tricine Acrylamide Gels
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Running Gel (10%)
- 4.9 mL 30% acrylamide/0.8% bisacrylamide (37.5:1)
- 5 mL Tris-Cl/SDS pH 8.45
- 1.15 mL MilliQ water
- 3.95 mL 40% glycerol
- 25 uL 10% APS (made fresh)
- 5 uL TEMED
Stacking Gel (4%)
- 0.81 mL 30% acrylamide/0.8% bisacrylamide
- 1.55 mL Tris-Cl/SDS pH 8.45
- 3.89 mL MilliQ water
- 40 uL saturated bromophenol blue (optional)
- 37.5 uL 10% APS (0.1g ammonium persulfate/mL H20)
- 17.5 uL TEMED
Add APS and TEMED immediately before pouring.
Pour running (separating) gel (use 20G 1.5 syringe) to 1 cm below bottom of comb (mark short plate) and overlay with isopropanol. Set for 1 hr, rinse with water (buffer might be better), and pour stacking gel. Insert combs, being careful to avoid bubbles. Let set, wrap in damp paper towels, and store 4 C for up to one week.
15 well, .75 mm gels hold about 15 uL sample per lane. To run gels (MiniProtean III), put 200 mL Anode Buffer in lower/outer chamber and 125 mL Cathode Buffer in inner/upper chamber. Remove combs after adding running buffers. Use a syringe or x-long gel-loading pipette tip to flush out each well gently with Cathode Buffer to remove any unpolymerized acrylamide.
Run at about 80 V for a couple of hours.
Tris-Tricine Cathode Buffer
- 12.22 g Tris (0.1 M)
- 17.92 g Tricine (0.1 M)
- 1 g SDS (0.1%)
- to 1 L with MilliQ H2O (no need to pH)
Tris-Tricine Anode Buffer (5L)
- 121 g Tris (0.2 M)
- 500 mL MilliQ H2O
- pH to 8.9 with 6N HCl (about 30 mL)
- to 5 L with MilliQ
Tris-Cl/SDS pH 8.45
- 182 g Tris base in 300 mL MilliQ H2O (Tris is 121.14 g/mol) (hard to dissolve)
- to pH 8.45 with 3N Hcl
- MilliQ H2O to 500 mL
- Filter 0.45 um
- Add 1.5 g SDS
