Hoefer SE640 - Manual (Page 40)

•
p32
6. 0.375 M TrisCl, 0.1% SDS, pH 8.8
(Resolving gel overlay, 100 ml)
1.5 M Tris-Cl, pH 8.8 (Soln. #2) 0.375 M 25.0 ml
10% SDS (Soln. #4)
3.5 mM 1.0 ml
Deionized H
2
O to 100.0 ml
—OR—
Water-saturated n-butanol
Shake n-butanol and deionized H
2
O in a separatory
funnel. Remove the aqueous (lower) phase. Repeat
this procedure several times. Use the upper phase.
—OR—
If an overlay interferes with the preferred protocol,
isolate the gel from atmospheric oxygen by placing a
blank comb or resolving gel former on the gel.
7. 2X Sample treatment buffer
(0.125 M TrisCl, 4% SDS, 20% glycerol,
2% 2-mercaptoethanol, pH 6.8, 10 ml)
0.5 M Tris-Cl, pH 6.8 (Soln. #3) 0.125 M 2.5 ml
10% SDS, 0.35 M (Soln. #4)
0.14 M 4.0 ml
Glycerol (FW 92.09) 20% v/v 2.0 ml
2-mercaptoethanol (FW 78.13) 2% v/v 0.2 ml
[–OR– Dithiothreitol (DTT) 0.2 mM 0.31 g]
(FW 154.2)
Bromophenol Blue (FW 691.9) 0.03 mM 0.2 mg
Deionized H
2
O to 10.0 ml
Divide into 1.0 ml aliquots and store at -40 °C to -80 °C.
—OR—
6X Sample treatment buffer
(0.35 M TrisCl, 10% SDS, 30% glycerol, 9.3% DTT,
pH 6.8, ~10 ml)
0.5 M Tris-Cl, pH 6.8 (Soln. #3) 0.35 M 7.0 ml
SDS (FW 288.4) 0.35 M 1.0 g
Glycerol (FW 92.09) 30% v/v 3.0 ml
DTT (FW 154.2) 0.6 M 0.93 g
Bromophenol Blue (FW 691.9) 0.175 mM 1.2 mg
Divide into 1.0 ml aliquots and store at -70 °C.