User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/14
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We will be using a Bio-Rad Mini Protean system with pre-cast Mini Protean TGX gels. The manual for this system can be found here We will be running SDS-PAGE followed by gel development with Coomassie Blue staining.
Below is a short description of how we will proceed. Please refer to the manual for more detailed instructions
-1mg BSA and 1mg Myoglobin in 1mL water -Solution was diluted to 1/10 to be used in gel
-14.4g Glycine, 1g SDS, 3g Tris in 100mL H2O -Solution was diluted to 1L with water
-40 Methanol, 10% Acetic Acid, 50% Water
-90% water, 10$ Acetic Acid and .0025% Commassie Brilliant Blue
-90% Water and 10% Acetic Acid
Cell Expression preparation
We are making more cells. 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 with 0.25g of NaCl (because broth mix did not contain any)
- For the 25mL LB Broth solution, 0.625g of LB Broth Media were dissolved into 25mL of water with 0.0625g of NaCl (because broth mix did not contain any)
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