User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/04

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Purpose

  • Grow and amplify ADA transfected BL21(DE3) E.coli cells in LB media\
  • Extract cells from media after ADA protein expression

Procedure

  • Centrifuge was turned on and pre-cooled to 4°C
  • 0.4M of Isopropyl β-D-1-thiogalactopyranoside (IPTG) in sterile water was calculated via the following way:
  Molecular weight: 238.31g/mol
  0.4M × 0.004L = 0.0016mol of IPTG needed
  238.31g/mol × 0.0016mol = 0.3813g of IPTG needed
  • 0.4M of IPTG was made in the following way:
    • 0.3813g of IPTG was weighted out
    • 4mL of autoclaved water was mixed with IPTG in a 15mL falcon tube
    • 1mL of 0.4M IPTG aliquot was made into 4 of 1.5mL microcentrifuge tubes for cell expression
  • Cells grew over night from 11/03/2012 in shaker were taken out. Four separate 35mL culture were poured into four separate 50mL falcon tube under sterile environment. Mass of the four cultures were balanced for successful centrifugation process. The four cultures were placed in centrifuge under 4°C, 4500rpm, for 15 minutes.
  • Orbital shaker was left on prewarmed to 37°C.
  • After centrifugation, pellets and supernatants form. The supernatants were discarded. The four pellets were resuspended with resuspending 4mL of autoclaved LB media in first pellet, then in the second pellet, third pellet, then fourth pellet. After 4 pellets from 4 different 50mL falcon tubes were resuspended, 1mL of resuspension were placed into each 1L autoclaved LB media from 11/03/2012.
  • Four aliquots of IPTG were added into each Fernbach flasks with cell culture before loaded into orbital shaker.
  • Cultures were left in orbital shaker for 4 hours, 210rpm, under 37°C.
  • After 4 hours of shaking, 1mL of cell were placed into a cuvette and run under UV-vis to test cell growth. The absorbance of cells in four Fernbach flasks were as follows:



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