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| ==Pouring Tris-Tricine Acrylamide Gels==
| | [[Endy:Tris-Tricine Acrylamide Gels]] |
| Protocol by [[Jennifer Braff|jcb]]
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| ==Running Gel (10%)==
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| *4.9 mL 30% acrylamide/0.8% bisacrylamide (37.5:1)
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| *5 mL Tris-Cl/SDS pH 8.45
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| *1.15 mL MilliQ water
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| *3.95 mL 40% glycerol
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| *25 uL 10% APS (made fresh)
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| *5 uL TEMED
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| ==Stacking Gel (4%)==
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| *0.81 mL 30% acrylamide/0.8% bisacrylamide
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| *1.55 mL Tris-Cl/SDS pH 8.45
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| *3.89 mL MilliQ water
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| *40 uL saturated bromophenol blue (optional)
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| *37.5 uL 10% APS (0.1g ammonium persulfate/mL H20)
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| *17.5 uL TEMED
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| Add APS and TEMED immediately before pouring.
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| 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.
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| 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.
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| Run at about 80 V for a couple of hours.
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| == Tris-Tricine Cathode Buffer==
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| *12.22 g Tris (0.1 M)
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| *17.92 g Tricine (0.1 M)
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| *1 g SDS (0.1%)
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| *to 1 L with MilliQ H2O (no need to pH)
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| == Tris-Tricine Anode Buffer (5L)==
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| *121 g Tris (0.2 M)
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| *500 mL MilliQ H2O
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| *pH to 8.9 with 6N HCl (about 30 mL)
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| *to 5 L with MilliQ
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| ==Tris-Cl/SDS pH 8.45==
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| *182 g Tris base in 300 mL MilliQ H2O (Tris is 121.14 g/mol) (hard to dissolve)
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| *to pH 8.45 with 3N Hcl
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| *MilliQ H2O to 500 mL
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| *Filter 0.45 um
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| *Add 1.5 g SDS
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