User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/04/08
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Dr Hartings' protocol can be found here.
SDS Page Gel Electrophoresis
I. 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.
II. 20 μL of each of the samples prepared yesterday, in addition to a ladder/marker, was loaded into one of four wells in the following order:
III. 4 - 12 well pre-cast Mini Protean TGX gel was obtained and prepared.
IV. The Bio-Rad Mini Protean system electrophoresis cell was assembled.
NOTE:The cell was initially prepared incorrectly, the "shorter wall" of the gel was supposed to face inside, not outside. The orientation of the gels was switched due to loading error and potentially contributed to the poor results.
V. The gel was run for approximately 40 min at 200 V, 0.05 A, and 10 W.
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.
VI. After 40 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 20 minutes
VII. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 20 minutes
VIII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) for 20 minutes and the results are pictured below:
NOTE: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.