Acrylamide gels

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 1: Line 1:
-
==Pouring Tris-Tricine Acrylamide Gels==
+
[[Endy:Tris-Tricine Acrylamide Gels]]
-
Protocol by [[Jennifer Braff|jcb]]
+
-
==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
+

Revision as of 11:05, 19 September 2005

Endy:Tris-Tricine Acrylamide Gels

Personal tools