User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2011/09/14

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Objective

Express the Maltose Binding Protein (MBP) in ER2566 E.coli with the pMAL-pIII plasmid.

Description

  1. The E.coli cultures grown overnight were centrifuged at 4500rpm for 15 minutes at 4°C.
  2. The supernatent was discarded and the cell pellets were each resuspended in 1mL of rich broth (4mL total).
  3. The resuspended pellets were evenly divided into four 2800mL flasks that contained 1L of rich broth with 100μg/mL ampicillin.
  4. These flasks were then shook at 165rpm at 37°C for about three hours. At this point 1mL of 0.1M IPTG was added to each flask.
  5. The cells continued to grow and now express protein for about three more hours at 165rpm at 37°C.
  6. The cultures were centrifuged at 4500rpm for 15 minutes at 4°C. In total the four pellets were resuspended in 200mL of column buffer. The cultures were then stored at -20°C.
  • Column Buffer: 20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA





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