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

 

■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 2

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■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