User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2013/02/06

Experimental Biological Chemistry II

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Purpose Remake LB+agar plates Make glycerol stock of DH10B and DH5α-T1 cells Test absorbance of cells in culture grew last night for cell concentration Procedure Because agar plates made on 2013/01/30 liquified, new plates were made. 12.5g of LB media powder were weighted and placed in a 750mL Erlenmeyer flask. 7.5g of Agar were weighted out and placed in the same 750mL Erlenmeyer flask. 500mL distilled water was measured out and placed in the same 750mL Erlenmeyer flask. The solution was autoclaved per liquid cycle. After autoclave, the agar were placed in room temperature to cool down to 60°C. Approximately 25mL of agar were poured into each petri dish. The agar plates were left in room temperature under flame to solidify. The agar plates were stored in fidge for future use. Glycerol stock of the two cell lines: DH10B and DH5α-T1 cells were made for future use. Two 5mL Gyro® Tubes were obtained, one for DH10B cells and another for DH5α-T1 cells. 150mL of autoclaved 60% glycerol in water was pipetted into the Gyro® Tubes prior. 850mL of DH10B cells, and 850mL of DH5α-T1 cells were pipetted into each of Gyro® Tubes, respectively. Each cell culture containing the cell and glycerol were vortexed until mix. Cells were placed in -80°C for storage. Absorbance of cells were taken using UV-vis spectroscopy to measure the concentration of cells that had been grown over night. UV-vis spectrometer was set under photometric method, with wavelength set at 650nm. A distilled water sample placed in plastic cuvette was placed into the spectrometer to eliminate background noise. 2mL of DH10B and DH5α-T1 cells were individually placed into a plastic cuvette into the spectrometer to measure absorbance. The absorbance for each cell after 15 hours of growth is shown in table below: Cells Absorbance DH10B 1.82 DH5α-T1 1.8 For future procedure on clay-incorporated silver, please refer to Melissa's Notebook