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

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:

• For future procedure on clay-incorporated silver, please refer to Melissa's Notebook