Manual

26
Dilute 200 ml of 5x buffer with 800 ml deionized water to prepare
1x electrophoresis buffer. The final concentrations of buffer
components will be.
pH Basic component Acidic component
3.8
30 mM
ß-Alanine
20 mM Lactic acid
4.4
80 mM
ß-Alanine
40 mM Acetic acid
4.8 80 mM GABA 20 mM Acetic Acid
6.1 30 mM Histidine 30 mM MES
6.6 25 mM Histidine 30 mM MOPS
7.4 43 mM Histidine 35 mM HEPES
8.1 32 mM Tris 30 mM EPPS
8.7 50 mM Tris 25 mM Boric acid
9.4 60 mM Tris 40 mM CHAPS
10.2 37 mM Ammonia 20 mM CAPS
Gel Formulations (10 ml)
1. Prepare the monomer solution by mixing all reagents except
the TEMED and 10% APS. Degas the mixture for 15 minutes.
30 % Degassed
Percent DDI H
2
O Acrylamide/Bis Gel buffer
gel (ml) (ml) (ml)
4% 6.7 1.3 2.0
5% 6.3 1.7 2.0
6% 6.05 2.0 2.0
Note: Prepare any desired volume of monomer solution by using
multiples of the 10 ml recipe.
2. Immediately prior to pouring the gel, for 10 ml monomer
solution add:
50 µl 10% APS
10 µl TEMED
Swirl gently to initiate polymerization.
Note: Below pH 6, TEMED becomes a less effective catalyst.
Increase the concentration of TEMED 5-fold to polymerize gels
with a pH range between 4 and 6.