User:Beatriz Gimenez De C./Notebook/572/2015/04/15

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Tasks

  • Run SDS-PAGE analysis with the solution prepared on Tuesday:

1. 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

2. 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.

3. 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

4. The gel was run for approximately 10 min at 200 V, 0.05 A, and 10 W.


5. After 10 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes

6. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for approximately 24 hours experiment continued tomorrow

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.


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