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| − | Phillip's notes
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| − | Introduction: Cells will be harvested, and cell lysis will be done if reagents are available.
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| − | Procedures:
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| − | Harvesting of cells
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| − | The 2L flask was taken out of the 30C incubator at 12:00PM, and left on the bench for about 2.5 hours. The cells appeared to be dense, as indicated by the milky-yellow color of the culture. Four 250mL centrifuge bottles were autoclaved, along with 1L of ddH2O. The rotor used for centrifugation was cooled in the cold room before use. Upon cooling the bottles and the sterile water, the liquid culture from the 2L flask was distributed evenly between three bottles (and balanced by adding sterile water). The bottles were centrifuged at 6000g for 15 minutes, and the supernatant was discarded. Each cell pellet was resuspended in 25mL of sterile water, and vortexed to resuspend. Each bottle was pooled, mixed, and distributed evenly between two 50mL falcon tubes. 8mL from one was taken to be used for a small-scale protein purification. The remaining bottles were balanced, and centrifuged again for 15min at 6000g. The supernatant was discarded, and the pellet was stored in the -20C freezer. The sample for small-scale purification was centrifuged, resuspended, and transferred to a 1.5mL before collecting the pellet to be frozen at -20C.
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| − | Conclusions: Since lysis cannot be continued until DNase, protease inhibitors, and lysozyme is obtained, it will be put on halt for the time being.
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