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

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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.