Team:TMU-Tokyo/Notebook/Assay 2 Protocol and Result

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<B>■Assay</B><Br>
<B>■Assay</B><Br>
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Device1 Assay<Br>
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<A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_0_Protocol_and_Result">Assay0</A><Br>
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<A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_1_Protocol_and_Result">Device1 Assay</A><Br>
Device2 Assay<Br>
Device2 Assay<Br>
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Device3 Assay<Br>
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<A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_3_Protocol_and_Result">Device3 Assay</A><Br>
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<Br>・Quantitative Analysis of Formaldehyde<Br>
<Br>・Quantitative Analysis of Formaldehyde<Br>
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ⅠPreparing a Standard Solution（Making a diluted solution of formaldehyde）<Br>
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Ⅰ Preparing a Standard Solution（Making a diluted solution of formaldehyde）<Br>
<Br>
<Br>
Add 1μl 20% formalin solution in DW399μl, diluted 400-fold. Make a 4000-fold dilution DW180μl take this and 20μl. I will continue to order from here a 2-fold dilution. This is the standard solution (diluted 4000,8000,16000,32000,64000,128000 fold dilution).<Br>
Add 1μl 20% formalin solution in DW399μl, diluted 400-fold. Make a 4000-fold dilution DW180μl take this and 20μl. I will continue to order from here a 2-fold dilution. This is the standard solution (diluted 4000,8000,16000,32000,64000,128000 fold dilution).<Br>
<Br>
<Br>
Ⅱ Making sample<Br>  
Ⅱ Making sample<Br>  
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①The cells were cultured at 30 ℃ for 18 hours in medium ampicillin resistance. Collected cells.<Br>
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1.The cells were cultured at 30 ℃ for 18 hours in medium<Br>
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②E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<Br>
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2.Centrifuged 10000 × g　5min<Br>
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③Centrifuged 10000 × 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)<Br>
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3.Remove the supernatant<Br>
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④Placed in an Eppendorf tube buffer and 2mM formaldehyde and 1 mM NAD ⁺ is coenzyme and  crude enzyme, it is allowed to react for 10 minutes at37 ℃.( Degrading enzyme from formaldehyde derived  in Pseudomonas. This enzyme decompose formaldehyde into formic acid in one minute 1.0umol in 1U(37 ℃ pH7.5))<Br>
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4.Wash the fungus<Br>
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⑤And this by the dilution of the sample corresponds to about 8000-fold dilution of 20% formaldehyde in  from 2mM buffer.<Br>
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Add dw and Centrifuged 10000 × g　5min<Br>
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5.Remove the supernatant<Br>
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6.E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<Br>
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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)<Br>
<Br>
<Br>
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ⅢCreating Standard Curves<Br>
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The reaction mixture are 50mM Tris-HCl ph7.2 0.8ml<Br>
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There formaldehyde 20mM 0.1ml (2mM final concentration)<Br>
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20mM NAD + 0.1ml<Br>
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Incubated for 5 minutes at 37 ℃ put<Br>
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I was allowed to react at 37 ℃ in the next, put 0.05ml crude enzyme solution.<Br>
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<Br>
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Ⅲ Creating Standard Curves and Measured samples<Br>
Liquid, the sample and standard experiment in triplicate. Performed only once blind<Br>
Liquid, the sample and standard experiment in triplicate. Performed only once blind<Br>
(40μl = 400μl for blind  = 360μl + 40μl × 3 × 3 lane:: each reagent = 120μl 40μl × 3 each consecutive sample)<Br>  
(40μl = 400μl for blind  = 360μl + 40μl × 3 × 3 lane:: each reagent = 120μl 40μl × 3 each consecutive sample)<Br>  
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<p class="description">
<p class="description">
<b>■Assay2 Result</b><Br>
<b>■Assay2 Result</b><Br>
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Standard Curves<Br>
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<Br>
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<IMG Src="https://static.igem.org/mediawiki/2012/a/a3/Assay2tmu.JPG" Width="640" Height="480"><Br>
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<Br>
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<b>concentration</b>
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<b>BBa_K749024</b><Br>
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<td>Enzyme+NAD</td>
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<td>NO enzyme</td>
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<td>NO NAD</td>
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<tr>
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<td>21.190</td>
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<td>25.629</td>
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<td>24.181</td>
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<Br>
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<Br>
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<b>wt</b><Br>
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<table border>
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<tr>
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<td>Enzyme+NAD</td>
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<td>NO enzyme</td>
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<td>NO NAD</td>
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</tr>
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<tr>
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<td>21.181</td>
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<td>26.984</td>
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<td>25.124</td>
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</tr>
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</table>
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<p class="description"><Br>All samples Reaction time 60min<Br>
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Latest revision as of 02:22, 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
Assay0
Device1 Assay
Device2 Assay
Device3 Assay




Assay 2


■Assay2 Protocol

・Quantitative Analysis of Formaldehyde

Ⅰ Preparing a Standard Solution（Making a diluted solution of formaldehyde）

Add 1μl 20% formalin solution in DW399μl, diluted 400-fold. Make a 4000-fold dilution DW180μl take this and 20μl. I will continue to order from here a 2-fold dilution. This is the standard solution (diluted 4000,8000,16000,32000,64000,128000 fold dilution).

Ⅱ Making sample
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 formaldehyde 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.

Ⅲ Creating Standard Curves and Measured samples
Liquid, the sample and standard experiment in triplicate. Performed only once blind
(40μl = 400μl for blind = 360μl + 40μl × 3 × 3 lane:: each reagent = 120μl 40μl × 3 each consecutive sample)
- The calibration curve
Absorbance = the absorbance of the standard solution- absorbance of the standard solution of blind accurate
①Into 40μl of each sample into a 96-well sample, standard, and blind.
Mix coloring reagent 40μl and alkaline reagent 40μl.
Allowed to stand at room temperature for 15 minutes at 20 ~ 35 ℃.
②Add 40μl reagent oxidation , (about 15 seconds) until the shaking stops foaming
③ Measured at 550nm wavelength in a microplate reader to measure the absorbance.

■Assay2 Result
Standard Curves



concentration
BBa_K749024

Enzyme+NAD NO enzyme NO NAD
21.190 25.629 24.181


wt
Enzyme+NAD NO enzyme NO NAD
21.181 26.984 25.124


All samples Reaction time 60min



 

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