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

From 2012.igem.org

(Difference between revisions)
 
(9 intermediate revisions not shown)
Line 3: Line 3:
<Html>
<Html>
 +
<Style Type="text/css">
 +
<!--
 +
td, th {
 +
  　　margin-left;
 +
  font: 11pt Verdana;
 +
  line-height: 1.5em;
 +
  letter-spacing: 0.06em;
 +
  color: black;
 +
 +
}
 +
-->
 +
</Style>
Line 31: Line 43:
<Br>
<Br>
<B>■Assay</B><Br>
<B>■Assay</B><Br>
-
Device1 Assay<Br>
+
<A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_0_Protocol_and_Result">Assay0</A><Br>
 +
<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>
-
Device3 Assay<Br>
+
<A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_3_Protocol_and_Result">Device3 Assay</A><Br>
Line 61: Line 74:
<Br>
<Br>
Ⅱ Making sample<Br>  
Ⅱ Making sample<Br>  
-
①The cells were cultured at 30 ℃ for 18 hours in medium ampicillin resistance. Collected cells.<Br>
+
1.The cells were cultured at 30 ℃ for 18 hours in medium<Br>
-
②E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<Br>
+
2.Centrifuged 10000 × g　5min<Br>
-
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>
+
3.Remove the supernatant<Br>
-
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>
+
4.Wash the fungus<Br>
-
⑤And this by the dilution of the sample corresponds to about 8000-fold dilution of 20% formaldehyde in  from 2mM buffer.<Br>
+
Add dw and Centrifuged 10000 × g　5min<Br>
 +
5.Remove the supernatant<Br>
 +
6.E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<Br>
 +
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>
-
Ⅲ Creating Standard Curves<Br>
+
 
 +
The reaction mixture are 50mM Tris-HCl ph7.2 0.8ml<Br>
 +
There formaldehyde 20mM 0.1ml (2mM final concentration)<Br>
 +
20mM NAD + 0.1ml<Br>
 +
Incubated for 5 minutes at 37 ℃ put<Br>
 +
I was allowed to react at 37 ℃ in the next, put 0.05ml crude enzyme solution.<Br>
 +
<Br>
 +
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>  
Line 81: Line 104:
<p class="description">
<p class="description">
<b>■Assay2 Result</b><Br>
<b>■Assay2 Result</b><Br>
 +
Standard Curves<Br>
 +
<Br>
 +
<IMG Src="https://static.igem.org/mediawiki/2012/a/a3/Assay2tmu.JPG" Width="640" Height="480"><Br>
 +
<Br>
 +
<b>concentration</b>
 +
 +
<Br>
 +
<b>BBa_K749024</b><Br>
 +
<table border>
 +
<tr>
 +
<td>Enzyme+NAD</td>
 +
<td>NO enzyme</td>
 +
<td>NO NAD</td>
 +
</tr>
 +
<tr>
 +
<td>21.190</td>
 +
<td>25.629</td>
 +
<td>24.181</td>
 +
</tr>
 +
</table>
 +
<Br>
 +
<Br>
 +
<b>wt</b><Br>
 +
<table border>
 +
<tr>
 +
<td>Enzyme+NAD</td>
 +
<td>NO enzyme</td>
 +
<td>NO NAD</td>
 +
</tr>
 +
<tr>
 +
<td>21.181</td>
 +
<td>26.984</td>
 +
<td>25.124</td>
 +
</tr>
 +
</table>
 +
<p class="description"><Br>All samples Reaction time 60min<Br>
</p>
</p>
<Br>
<Br>

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



 

Retrieved from "http://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_2_Protocol_and_Result"