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 UV-1800 UV-vis spectrometer from Shimadzu was used to measure absorbance via the following settings:
  Wavelength: 600nm
  Settings: Photometric
  Data display: Absorbance
  • Centrifuge was turned out to pre-cool to 4°C.
  • The absorbance of cells in four Fernbach flasks were as follows:
Flask Volume of cell culture[mL] Wavelength[nm] Absorbance
126000.683
226000.691
326000.657
426000.701
  • Cell culture were taken out of orbital shaker and transferred into centrifuge bottles. Mass of centrifuge bottles were balanced out to make sure successful centrifugation. The cell culture were placed into centrifuge pre-cooled to 4°C and spin for 30 minutes, 4500rpm, at 4°C.
  • Supernatants from centrifuged products were discarded. All four pellets from four centrifuge bottles were resuspended in 30mL binding buffer made on 09/26/2012 with the following condition:
  20mM Tris buffer
  0.5M NaCl
  30mM Imidizole
  pH 7.5
  • Cells from pellets were resuspended in the following way: 30mL of binding buffer was first placed in first centrifuge bottle with pellet, cells resuspended using pipetman and vortexer. After cells were completely resuspended in first bottle, the culture were transfered into second centrifuge bottle. This process was repeated until culture containing binding buffer and suspended cell pellets from first, second, third, and fourth centrifuge bottle were well mixed.
  • Pellet with binding buffer resuspension were stored in a 50mL falcon tube, and stored in freezer at -80°C.

Notes

  • IPTG was added to induce cell expression. IPTG was added immediately before cells were placed in orbital shaker to make sure that cells were under ideal conditions when expression proteins.
  • The absorbance for cells after 4 hours of growth were higher than expected (an absorbance of 0.600 at 600nm). This indicates the cells were overgrown. The cells were harvested nonetheless. The high absorbance indicates more cells in flasks, thus more protein when purified.
  • The cell cultures were centrifuged for 30 minutes as oppose to the protocol which suggests 15 minutes. Longer centrifugation time is used here to make sure all cells were in the pellet.
  • The final volume of cell resuspension is 42mL due to the addition of cell pellet as well as small amounts of supernatant that were not completely cleared out.


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