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Team:TMU-Tokyo/Notebook/Assay 3 Protocol and Result
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<b>■Assay3 Result</b><Br> | <b>■Assay3 Result</b><Br> | ||
<Br> | <Br> | ||
| + | only formate 2mM<Br> | ||
| + | Peak = 8.81(min)<Br> | ||
| + | |||
<b>BBa_K749015</b> | <b>BBa_K749015</b> | ||
| - | <Br> | + | <Br> |
| - | + | <Br> | |
| - | + | <Br> | |
| - | + | <p class="description"> | |
| + | <table border> | ||
| + | <tr> | ||
| + | <td><p class="description"> Reaction time </td> | ||
| + | <td> volume </td> | ||
| + | <td> Peak area </td> | ||
| + | <td> Peak area(%) </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>30min </td> | ||
| + | <td>50μl</td> | ||
| + | <td>229371.2</td> | ||
| + | <td>80.88</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10min</td> | ||
| + | <td>20μl</td> | ||
| + | <td>248294.2</td> | ||
| + | <td>83.9823</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
| + | <Br> | ||
| + | <Br> | ||
<b>WT</b><Br> | <b>WT</b><Br> | ||
10min 20μl 232791.6 83.691<Br> | 10min 20μl 232791.6 83.691<Br> | ||
| - | + | <b>No enzym</b><Br> | |
| + | 10min 243418.6 84.37<Br> | ||
</p> | </p> | ||
<Br> | <Br> | ||
Revision as of 01:56, 27 September 2012
■Experiment
1st week (8.13 - 8.19)
2nd week (8.20 - 8.26)
3rd week (8.27 - 9. 2)
4th week (9. 3 - 9. 9)
5th week (9.10 - 9.16)
6th week (9.17 - 9.23)
7th week (9.24 - 9.30)
■Protocols
■Assay
Device1 Assay
Device2 Assay
Device3 Assay
Assay 3
■Assay3 Protocol
Ⅰ.Extraction of the crude enzyme solution
1.The cells were cultured at 30 ℃ for 18 hours in medium
2.Centrifuged 10000 × g 5min
3.Remove the supernatant
4.Wash the fungus
Add dw and Centrifuged 10000 × g 5min
5.Remove the supernatant
6.E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .
7.Centrifuged 11000 × g at 4 ℃ so that the enzyme is not deactivated, the supernatant was harvested as crude enzyme solution (If you want to save is stored at 4 ℃ in a cold room)
The reaction mixture are
50mM Tris-HCl ph7.2 0.8ml
There sodium formate 20mM 0.1ml (2mM final concentration)
20mM NAD + 0.1ml
Incubated for 5 minutes at 37 ℃ put
I was allowed to react at 37 ℃ in the next, put 0.05ml crude enzyme solution.
Formic acid formic acid was quantified through the reaction mixture liquid chromatography.
■Assay3 Result
only formate 2mM
Peak = 8.81(min)
BBa_K749015
Reaction time |
volume | Peak area | Peak area(%) |
| 30min | 50μl | 229371.2 | 80.88 |
| 10min | 20μl | 248294.2 | 83.9823 |
WT
10min 20μl 232791.6 83.691
No enzym
10min 243418.6 84.37
