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

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Contents

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.
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