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More ADA and AAS
Goals
- Run AAS from Au/BSA solutions
- Make starter culture media and expression culture media in order to produce more ADA protein.
Procedure
- For the Starter culture media and expression culture media, the same procedure from September 18
- The samples of broth were made and autoclaved today. The antibiotic and the E.coli colony were also added into the starter culture media.
- The LB was weighed out into 4 large Fernbach flasks and 4 small erlyenmeyer flasks. They were capped with aluminum foil and placed in the Autoclave at a temperature of 121°C for 60minutes followed by a 30 minute cool down period. The autoclave is necessary in order to be sterilized and to ensure that no microbes from the surrounding environment could compete with the bacteria. After the first four samples were removed from the autoclave, the remaining 4 were placed in the autoclave for 50 minutes at the same temperature and were cooled down for 30 minutes as well. The samples had to be autoclaved separately because of lack of room in the autoclave.
- After the flasks had cooled, .035mL of Kanamycin was added to each of the starter culture flask.
- The bacteria was inoculated by retrieving a single colony from the petri dish and dropping it into the flask.
- After all the starter culture media broths cooled down, they were inoculate with transformed Escherichia coli. Aseptic techniques are implemented to prevent any undesirable microbes from being included in the inoculation of the media.
- The starter culture media flasks were then placed in a shaker incubator at 37°C at 200rpm overnight.
- For AAS: The 14 samples produced fibers this time and as a result, the fibers needed to be removed which was done through centrifuging and then pipetting out the solution without the fibers. For everything done in regards to the AAS today please see Mary's notebook.
Data
Starter Culture Media-- amount of LB added to each flask
- Flask 1: 25.0095g
- Flask 2: 25.0059g
- Flask 3: 25.009g
- Flask 4: 25.0008g
Expression Culture Media:
- Flask 1: .8744g
- Flask 2: .8775g
- Flask 3: .8745g
- Flask 4L .8749g
| Standard
|
Concentration
|
Absorbance
|
Background
|
| 5 |
- |
0.1132 |
-0.0057
|
| 8 |
- |
0.1835 |
-0.001
|
| 10 |
- |
0.2205 |
0.0008
|
| 15 |
- |
0.3209 |
-0.0018
|
| 20 |
- |
0.4288 |
0.0033
|
| 25 |
- |
0.5279 |
0.0043
|
| 30 |
- |
0.5989 |
0.0062
|
| 40 |
- |
0.7758 |
0.0052
|
| Blank |
- |
0.0134 |
0.009
|
|
|
| Sample
|
Concentration
|
Absorbance
|
Background
|
| 60 |
10.2247 |
0.2274 |
0.0055
|
| 80 |
16.1569 |
0.3399 |
0.0071
|
| 100 |
-1.1125 |
0.0124 |
-0.0008
|
| 120 |
21.256 |
0.4366 |
0.0073
|
| 128 |
-1.2496 |
0.0098 |
0.0002
|
| 130 |
-0.8858 |
0.0167 |
0.0029
|
| 132 |
-1.3973 |
0.007 |
0.0036
|
| 133 |
-1.2074 |
0.0106 |
0.0034
|
| 134 |
-1.3024 |
0.0088 |
0.0014
|
| 136 |
-1.4447 |
0.0061 |
0.003
|
| 138 |
-1.1073 |
0.0125 |
-0.0002
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| 140 |
-1.2338 |
0.0101 |
0.0028
|
| 160 |
-1.0387 |
0.0138 |
0.002
|
| 170 |
-0.0969 |
0.0163 |
0.002
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Calculations of comparisons between measured gold concentration and actual concentration after the removal of the fibers from the solutions to come in the future.
File:AAS data.xlsx
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