User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/01/31

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Objective

Transform the mutated GFP into an expression strain of E. coli. Prepare the amylose resin for purification. Prepare media for E.coli expression.

Description

Transformation

  1. Mix 30μL of BL21(DE3) Competent E.coli with 5μL of the mutated GFP in the cold, sterile tube.
  2. Incubate this mixture for 30 minutes on ice.
  3. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
  4. Add 250μL of SOC media to the cells/PCR product.
  5. Shake the mixture at 250rpm at 37°C for one hour.
  6. Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin.
    • The LB plate was made with 0.875g LB, 0.7g Agar, and 35mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 35μL of 100mg/mL ampicillin was added to it and it was poured into a sterile petri dish.
  7. Incubate the plate overnight (inverted) at 37°C.

Regeneration of Amylose Resin Column

  1. Run 90mL of distilled water over the column at 4mL/min.
  2. Run 90mL of 0.1% SDS over the column at 4mL/min.
  3. Run 30mL of distilled water over the column at 4mL/min.
  4. Run 150mL of column buffer (20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA) over the column at 4mL/min.

Preparation of Media for Expression

  1. Combine 25g LB and 1L of distilled water in a 2.8L flask. (2 flasks)
  2. Autoclave the flasks (liquid cycle).

Data

  • The plate that was incubated overnight (from the GFP transformation) grew many colonies. The colonies had a light green color.




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