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Team:TMU-Tokyo/Notebook/Assay 2 Protocol and Result
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| - | Assay | + | Assay 2 |
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<b>■Assay2 Protocol</b><Br> | <b>■Assay2 Protocol</b><Br> | ||
| - | <Br><Br></p> | + | <Br>・Quantitative Analysis of Formaldehyde<Br> |
| + | <Br> | ||
| + | ⅠPreparing a Standard Solution（Making a diluted solution of formaldehyde）<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> | ||
| + | <Br> | ||
| + | Ⅱ Making sample<Br> | ||
| + | ①The cells were cultured at 30 ℃ for 18 hours in medium ampicillin resistance. Collected cells.<Br> | ||
| + | ②E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<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> | ||
| + | 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> | ||
| + | ⑤And this by the dilution of the sample corresponds to about 8000-fold dilution of 20% formaldehyde in from 2mM buffer.<Br> | ||
| + | <Br> | ||
| + | ⅢCreating Standard Curves<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> | ||
| + | - The calibration curve <Br> | ||
| + | Absorbance = the absorbance of the standard solution- absorbance of the standard solution of blind accurate<Br> | ||
| + | |||
| + | ①Into 40μl of each sample into a 96-well sample, standard, and blind.<Br> | ||
| + | Mix coloring reagent 40μl and alkaline reagent 40μl.<Br> | ||
| + | Allowed to stand at room temperature for 15 minutes at 20 ~ 35 ℃.<Br> | ||
| + | ②Add 40μl reagent oxidation , (about 15 seconds) until the shaking stops foaming<Br> | ||
| + | ③ Measured at 550nm wavelength in a microplate reader to measure the absorbance.<Br> | ||
| + | <Br></p> | ||
<p class="description"> | <p class="description"> | ||
<b>■Assay2 Result</b><Br> | <b>■Assay2 Result</b><Br> | ||
Revision as of 07:56, 26 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 2
■Assay2 Protocol
・Quantitative Analysis of Formaldehyde
ⅠPreparing 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.
ⅢCreating 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
