Want |
Reagent |
[stock]/FW |
For 20mL |
125 mM |
Phosphate Buffer pH 7.0 |
1M |
2.5 mL |
200 mM |
DTT |
|
0.31 g |
20% |
Glycerol (Anal-R) |
|
4 mL |
4% SDS |
SDS |
10% |
8 mL |
0.02% |
Bromophenol Blue |
|
A few crystals |
|
ddH2O |
|
Fill to 20mL |
Want |
Reagent |
For 2L |
250mM |
Tris Base |
60g |
2M |
Glycine |
280g |
|
SDS |
20g |
- fill to 2L with ddH2O
Want |
Reagent |
For 2L |
250mM |
Tris (not pH’d) |
60.6g |
2M |
Glycine |
300.3g |
- fill to 2L with ddH2O
- to use, dilute in 20% Methanol (in ddH2O)
Buffer B + 3%w/v skim milk powder
Want |
Reagent |
For 1L |
250mM |
Tris-HCl pH 7.5 |
30.23g |
750mM |
NaCl |
43.33g |
0.25% |
Tween-20 |
2.5mL |
0.2% |
NaN3 |
2g |
- fill to 1L with ddH2O, pH the solution with HCl
- Buffer A is this plus 3% skim milk powder
Want |
Reagent |
[stock]/FW |
For 500 mL |
100mM |
Tris-HCl pH 9.5 |
|
6.055g |
100mM |
NaCl |
|
2.022g |
2mM |
MgCl2 |
3M |
0.5mL |
0.05% |
NP40 or Tween-20 |
|
0.25mL |
To use: 33uL 50mg/ml BCIP (in 100% DMF) + 66uL 50mg/ml NBT (in 70% DMF) + 10mL Buffer C
Target Acrylamide Concentration |
7.5% |
9% |
10% |
10.5% |
11% |
12% |
ddH2O |
4.8 mL |
4.35 mL |
4.0 mL |
3.9 mL |
3.7 mL |
3.4 mL |
30% Acrylamide |
2.5 mL |
3.0 mL |
3.3 mL |
3.5 mL |
3.7 mL |
4.0 mL |
1.5M Tris-HCl (pH8.8) |
2.5 mL |
2.5 mL |
2.5 mL |
2.5 mL |
2.5 mL |
2.5 mL |
10% SDS |
100 mL |
100 mL |
100 mL |
100 mL |
100 mL |
100 mL |
10% APS |
50 mL |
50 mL |
50 mL |
50 mL |
50 mL |
50 mL |
TEMED |
10 mL |
10 mL |
10 mL |
10 mL |
10 mL |
10 mL |
*Add APS and TEMED last after degassing.
Acrylamide |
4% |
ddH2O |
7.35 mL |
30% Acrylamide |
1.3 mL |
1M Tris-HCl (pH6.8) |
1.25 mL |
10% SDS |
100 mL |
10% APS |
50 mL |
TEMED |
10 mL |