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

■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
①The cells were cultured at 30 ℃ for 18 hours in medium ampicillin resistance. Collected cells.
②E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .
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)
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))
⑤And this by the dilution of the sample corresponds to about 8000-fold dilution of 20% formaldehyde in from 2mM buffer.

Ⅲ Creating Standard Curves
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
concentration

All samples Reaction time 60min
BBa_K749024
Enzyme+NAD 21.190
NO enzyme 25.629
NO NAD 24.181

wt
Enzyme+NAD 21.181
NO enzyme 26.984
NO NAD 25.124