User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/25: Difference between revisions
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==Filtration of Buffers== | ==Filtration of Buffers== | ||
[[Image:Bufpix.jpg|thumb|right|Assembly of Membrane Filtration Image Source: http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/counting.html]] | |||
* The assembly shown in the picture on the right was followed with an additional clip that would hold the filter tightly to the flask. The sidearm of the 1000 mL flask was connected to a Welch vacuum pump. | |||
* The membrane filter had a diameter of 47mm with a 450 nm pore diameter. | |||
* The pump was turned on; the knob was unscrewed. The setting was set to 60 cmHg vacuum. This setting was chosen at random. | |||
* The first pour was done with the binding buffer. This was done to ensure that the buffer with the lower imidazole content (binding) would not be contaminated with the buffer that had the higher imidazole content (elution). | |||
* The collected filtrate of the binding buffer was poured into a sterilized glass bottle. This was followed by the pouring of the elution buffer into the filtering system. The filtrate for the elution buffer was also poured into a sterilized glass bottle. | |||
* The bottles were capped with aluminum foil tops and refrigerated. The buffers were clear, colorless liquid solution before and after filtration. | |||
* The filtrates were poured into their respective falcon tubes and refrigerated. | |||
==Filtration of the Supernatant== | |||
* The centrifuge tubes were taken out from the chamber. The supernatant of the tubes were poured into the filtering system. Sample 1 remained isolated from sample 2 which contained pellets 2, 3, and 4 from last week. | |||
* The current pellet was left untouched in the centrifuge tubes. This are discarded material that contains disrupted cell organelles and other cellular material. | |||
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Revision as of 06:39, 7 December 2012
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Lysis of transformed E. coli
Filtration of Buffers
Filtration of the Supernatant
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