User:Beatriz Gimenez De C./Notebook/572/2015/04/08

Tasks

• Perform SDS Page Gel Electrophoresis with solutions made on Tuesday
  • A 10x SDS-PAGE Running Buffer (30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) was diluted by a factor of 10.
    • 100 mL of the 10x buffer was added to a 1000 mL volumetric flask
  • load on wells 20 μL of each of the samples prepared yesterday, headed for 5 min on thermocylender + a ladder/marker in the following order:

Gel AO

Well	Sample
1	Ladder
2	1 μM Proteinase K - Dilute
3	100 nM Proteinase K - Dilute
4	10 nM Proteinase K - Dilute
5	1 nM Proteinase K - Dilute
6	Blank
7	1 μM Proteinase K
8	100 nM Proteinase K
9	10 nM Proteinase K
10	1 nM Proteinase K

Gel AI

Well	Sample
1	Ladder
2	Blank
3	1 μM Trypsin - Dilute
4	100 nM Trypsin - Dilute
5	10 nM Trypsin - Dilute
6	1 nM Trypsin - Dilute
7	Blank
8	1 μM Trypsin
9	100 nM Trypsin
10	10 nM Trypsin
11	1 nM Trypsin

Gel BI

Well	Sample
1	Ladder
2	Blank
3	1 μM Chymotrypsin - Dilute
4	100 nM Chymotrypsin - Dilute
5	10 nM Chymotrypsin - Dilute
6	1 nM Chymotrypsin - Dilute
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 - Dilute
4	100 nM Thermolysin - Dilute
5	10 nM Thermolysin - Dilute
6	1 nM Thermolysin - Dilute
7	Blank
8	1 μM Thermolysin
9	100 nM Thermolysin
10	10 nM Thermolysin
11	1 nM Thermolysin

• 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
• 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.
• The gel was run for approximately 40 min at 200 V, 0.05 A, and 10 W.
• After 40 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 20 minutes
• The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 20 minutes
• Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) for 20 minutes and the results are pictured below:

Results

NOTE

• Only the B side gel appeared to be running for the first approx. 20 minutes; adjustments were made but the A side gels ultimately only ran for about 20 min instead of the suggested 40.
• The fixing, staining, and de-staining processes were shortened because of time limitations. Additionally, the results demonstrate what appears to be primarily protein ladder/marker that made its way into adjacent wells, and nothing else.