User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/04/08: Difference between revisions
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Dr Hartings' protocol can be found [http://openwetware.org/wiki/User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/25 here]. | Dr Hartings' protocol can be found [http://openwetware.org/wiki/User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/25 here]. | ||
==April 8<sup>th</sup>== | |||
=='''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. | |||
*100 mL of the 10x buffer was added to a 1000 mL volumetric flask | |||
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: | |||
*The samples were heated for 5 min at 100C in the thermocycler pre-loading | |||
'''Gel AO''' | |||
==<font color=#0f1028></font>== | |||
{|style="width:700px" | |||
|<u>'''Well'''</u> | |||
|<u>'''Sample'''</u> | |||
|- | |||
|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''' | |||
==<font color=#0f1028></font>== | |||
{|style="width:700px" | |||
|<u>'''Well'''</u> | |||
|<u>'''Sample'''</u> | |||
|- | |||
|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''' | |||
==<font color=#0f1028></font>== | |||
{|style="width:700px" | |||
|<u>'''Well'''</u> | |||
|<u>'''Sample'''</u> | |||
|- | |||
|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''' | |||
==<font color=#0f1028></font>== | |||
{|style="width:700px" | |||
|<u>'''Well'''</u> | |||
|<u>'''Sample'''</u> | |||
|- | |||
|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 | |||
|- | |||
|} | |||
III. 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 | |||
IV. 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. | |||
'''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. | |||
[[Image:IMG_4314.JPG|500px]] | |||
[[Image:IMG_4315.JPG|500px]] | |||
[[Image:IMG_4316.JPG|500px]] | |||
[[Image:IMG_4317.JPG|500px]] | |||
Revision as of 13:14, 18 April 2015
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Running SDS-PAGEDr Hartings' protocol can be found here. April 8thSDS Page Gel ElectrophoresisI. 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:
Gel AO
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.
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