User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/04: Difference between revisions
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* 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. | * 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 with the following condition: | * Supernatants from centrifuged products were discarded. All four pellets from four centrifuge bottles were resuspended in 30mL binding buffer made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/25|10/25/2012]] with the following condition: | ||
20mM Tris buffer | 20mM Tris buffer | ||
0.5M NaCl | 0.5M NaCl | ||
30mM Imidizole | 30mM Imidizole | ||
pH 7.5 | 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. | * Pellet with binding buffer resuspension were stored in a 50mL falcon tube, and stored in freezer at -80°C. | ||
Revision as of 17:19, 4 November 2012
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Purpose
Procedure
Molecular weight: 238.31g/mol 0.4M × 0.004L = 0.0016mol of IPTG needed 238.31g/mol × 0.0016mol = 0.3813g of IPTG needed
Wavelength: 600nm Settings: Photometric Data display: Absorbance
20mM Tris buffer 0.5M NaCl 30mM Imidizole pH 7.5
Notes
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