User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/06/02

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Preparing Broth

Objective

During this lab session, solutions LB Broth solutions were prepared. A solution of Ampicilin was prepared to be added to the broth and agar.

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

Protein purification

The same method was continued to attempt to purify the extracted proteins It seems that the protein solutions come out cloudy and a precipitated forms at the bottom. The solution's pH was adjusted to ~9 using 1M NaOH, the solution remained cloudy After two hours n the bench, the solution was spun at 20,000rpm for 2 hours at 4 degrees Celsius -It seems that the original solution has changed

  1. before it was clear with a reddish tint
  2. Now it is cloudy, with precipitate forming and the solution is no longer red but brown


Preparations for tomorrow

4L of 50mM Tris buffer was prepared at pH 9 -24.228g Tris in 4L H2O. pH adjusted to 9 using 1N HCl


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