User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/26: Difference between revisions
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* In the morning, cells left in shaker were taken out and spin in centrifuge at 4500rpm for 15 minutes. Pellets form, and supernatant were discarded. Pellet were resuspended in 4mL of LB. 1mL of each cell culture were placed in 1L autoclaved sterile LB broth, 1mL of 0.4M IPTG were added before incubation. Cell culture were placed in shaker under 225rpm at 37C for ~4 hours. | * In the morning, cells left in shaker were taken out and spin in centrifuge at 4500rpm for 15 minutes. Pellets form, and supernatant were discarded. Pellet were resuspended in 4mL of LB. 1mL of each cell culture were placed in 1L autoclaved sterile LB broth, 1mL of 0.4M IPTG were added before incubation. Cell culture were placed in shaker under 225rpm at 37C for ~4 hours. | ||
* Elution and binding buffers were made. See [[User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/26|Dhea's Notebook]] for the details in making elution and binding buffers used for future cell resuspension and protein purification. | * Elution and binding buffers were made. See [[User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/26|Dhea's Notebook]] for the details in making elution and binding buffers used for future cell resuspension and protein purification. | ||
* After 4 hours in the shaker, cells were transferred into centrifuge bottles with mass ~1 grams apart. Cells were centrifuged at 4500rpm for 15 minutes at 4C. Supernatant were discarded, pellet were resuspended with 30mL of binding buffer. Cell resuspension were stored in -80C until next week. This process is repeated two times for more cells. | |||
* After 4 hours in the shaker, cells were transferred into centrifuge bottles with mass ~1 grams apart. Cells were centrifuged at 4500rpm for 15 minutes at 4C. Supernatant were discarded, pellet were resuspended with 30mL of binding buffer. Cell resuspension were stored in -80C until next week. | * Luminol-peroxide reaction were further experimented. Luminol concentration were altered, and concentration of horseradish peroxidase were altered. For details of the experience, and details of data obtained, see [[User:Melissa Novy/Notebook/CHEM-571/2012/09/26|Melissa's Notebook]] for information on concentration of luminol, H2O2, and HRP assay used in each trial. For initial and final concentration of the solutions, see below table: | ||
{| {{table}} | |||
* Luminol-peroxide reaction were further | | align="center" style="background:#f0f0f0;"|'''''' | ||
| align="center" style="background:#f0f0f0;"|'''Initial Concentrations [mM]''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Final Concentrations [mM]''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| Trial||4-Iodophenol||Luminol||H<sub>2</sub>O<sub>2</sub>||HRP||4-Iodophenol||Luminol||H<sub>2</sub>O<sub>2</sub>||HRP | |||
|- | |||
| 1||18||1.25||1.7||0.0023||0.000119||0.000578||0.000846||7.63E-08 | |||
|- | |||
| 2||18||0.625||1.7||0.0023||0.000119||0.000289||0.000846||7.63E-08 | |||
|- | |||
| 3||18||0.625||0.85||0.0023||0.000119||0.000289||0.000423||7.63E-08 | |||
|- | |||
| 4||18||1.25||0.425||0.0023||0.000119||0.000578||0.000212||7.63E-08 | |||
|- | |||
| 5||18||1.25||1.7||0.0023||0.000119||0.000578||0.000846||7.63E-08 | |||
|- | |||
| 6||18||1.25||1.7||0.00115||0.000119||0.000578||0.000846||3.82E-08 | |||
|} | |||
==Notes== | ==Notes== |
Revision as of 20:34, 26 October 2012
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Purpose
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