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

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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


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