User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/04/15: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->=='''SDS Page Gel Electrophoresis'''== | ||
== | |||
I. 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 | |||
II. 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. | |||
III. 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''' | |||
==<font color=#0f1028></font>== | |||
{|style="width:700px" | |||
|<u>'''Well'''</u> | |||
|<u>'''Sample'''</u> | |||
|- | |||
|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''' | |||
==<font color=#0f1028></font>== | |||
{|style="width:700px" | |||
|<u>'''Well'''</u> | |||
|<u>'''Sample'''</u> | |||
|- | |||
|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''' | |||
==<font color=#0f1028></font>== | |||
{|style="width:700px" | |||
|<u>'''Well'''</u> | |||
|<u>'''Sample'''</u> | |||
|- | |||
|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''' | |||
==<font color=#0f1028></font>== | |||
{|style="width:700px" | |||
|<u>'''Well'''</u> | |||
|<u>'''Sample'''</u> | |||
|- | |||
|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 | |||
|- | |||
|} | |||
IV. The gel was run for approximately 10 min at 200 V, 0.05 A, and 10 W. | |||
'''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. | |||
V. After 10 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes | |||
VI. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for approximately 24 hours | |||
VII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) three times for 15 minutes each and the following results were obtained: | |||
[[Image:11134235_10205059533466235_680314354_n_%281%29.jpg|500px]] | |||
[[Image:11149169_10205059536186303_2003984602_n.jpg|500px]] | |||
[[Image:11123991_10205059535426284_1373617103_n.jpg|500px]] | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Latest revision as of 00:55, 27 September 2017
Project name | Main project page Previous entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SDS Page Gel ElectrophoresisI. 4 - 12 well pre-cast Mini Protean TGX gel was obtained and prepared.
II. The Bio-Rad Mini Protean system electrophoresis cell was assembled.
III. Each of the 4 gels were loaded with each of the 20 μL reaction samples prepared yesterday in the following manner:
IV. The gel was run for approximately 10 min at 200 V, 0.05 A, and 10 W. 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. V. After 10 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes VI. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for approximately 24 hours VII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) three times for 15 minutes each and the following results were obtained: |