Difference between revisions of "Acrylamide gels"

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==Pouring Tris-Tricine Acrylamide Gels==
==Lab Protocols==
==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)
[[Endy:Tris-Tricine Acrylamide Gels]]<br>
[[Sauer:bis-Tris SDS-PAGE, the very best]]<br>
==Stacking Gel (4%)==
[[Keating:Experimental Protocols:SDS-PAGE]]<br>
*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. 
[[Category:In vitro]]
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

Latest revision as of 13:10, 10 August 2006

Lab Protocols

Endy:Tris-Tricine Acrylamide Gels
Sauer:bis-Tris SDS-PAGE, the very best
Keating:Experimental Protocols:SDS-PAGE