User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/08

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Protein Purification

  1. The protein in phosphate buffer were placed in the centrifuge again to be concentrated with more pH7.2 phosphate buffer
  2. This solution was run through the SP-Sepharose column
    1. It appeared that nothing stuck to the column and the elute was collected
  3. The column was eluted with a linear gradient (0-100%) of pH8 phosphate buffer
    1. Nothing seemed to come off

Cell Expression

Procedure

1. The correct concentration for dissolving LB Broth Media into water is 25g per liter of water.

- For the 1L LB Broth solution, 25g of LB Broth Media were dissolved into 1L of water

- For the 25mL LB Broth solution, 0.625g of LB Broth Media were dissolved into 25mL of water

The solution are placed in the autoclave at 121 degrees C for an hour.

2. In order to prepare the ampicilin solution, 500mg of Ampicilin Sodium Salt were dissolved into 5mL of sterilized water. This solution was prepared in a sterilized eppendorf tube using sterile equipment.

3. Once the solutions cool, 25uL of the ampicilin solution is added to each of the 25ml flasks and 1mL is added to each of the 1L flasks

4. The 25mL flasks were inoculated with K31c Asc Hb (BL21DE3) cells and placed on the shaking incubator at 37C and 225rpm overnight


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