Tasks
- Run SDS-PAGE analysis with the solution prepared on Tuesday
- Staining was not complete so had to keep gels submerged in staining solution over night.
I. 4 - 12 well pre-cast Mini Protean TGX gel was obtained and prepared.
- The comb and tape was removed from each gel
- The wells of each gel were rinsed with the prepared running buffer dilution
II. The Bio-Rad Mini Protean system electrophoresis cell was assembled.
- The inner and outer buffer chambers were filled with the diluted running buffer to the "4 gel" mark.
III. Each of the 4 gels were loaded with each of the 20 μL reaction samples prepared yesterday in the following manner:
- The samples were heated for 5 min at 100C in the thermocycler pre-loading
Gel AO
| Well
|
Sample
|
| 1
|
Ladder
|
| 2
|
BLANK
|
| 3
|
1 μM Proteinase K (I)
|
| 4
|
100 nM Proteinase K (I)
|
| 5
|
10 nM Proteinase K (I)
|
| 6
|
1 nM Proteinase K (I)
|
Gel AI
| Well
|
Sample
|
| 1
|
Ladder
|
| 2
|
BLANK
|
| 3
|
1 μM Proteinase K (II)
|
| 4
|
BLANK
|
| 5
|
100 nM Proteinase K (II)
|
| 6
|
BLANK
|
| 7
|
10 nM Proteinase K (II)
|
| 8
|
BLANK
|
| 9
|
1 nM Proteinase K (II)
|
Gel BI
| Well
|
Sample
|
| 1
|
Ladder
|
| 2
|
BLANK
|
| 3
|
1 μM Trypsin
|
| 4
|
100 nM Trypsin
|
| 5
|
10 nM Trypsin
|
| 6
|
1 nM Trypsin
|
| 7
|
BLANK
|
| 8
|
1 μM Chymotrypsin
|
| 9
|
100 nM Chymotrypsin
|
| 10
|
10 nM Chymotrypsin
|
| 11
|
1 nM Chymotrypsin
|
Gel BO
| Well
|
Sample
|
| 1
|
Ladder
|
| 2
|
BLANK
|
| 3
|
1 μM Thermolysin
|
| 4
|
SKIP
|
| 5
|
100 nM Thermolysin
|
| 6
|
SKIP
|
| 7
|
10 nM Thermolysin
|
| 8
|
BLANK
|
| 9
|
1 nM Thermolysin
|
IV. The gel was run for approximately 10 min at 200 V, 0.05 A, and 10 W.
V. After 10 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
VI. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for approximately 24 hours
Note
- The top of the chamber was not functioning properly in that a closed circuit was not established and the top had to be manually held/pushed down for the entirety of the run.
| 
Project name
