User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/02/15

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Objective

Prepare for GFP purification. Prepare for a MBP-intein expression next week.

Description

GFP Purification Prep

  1. Sterile filter the GFP lysate into 50mL sterile Falcon tubes.
  2. Prepare a binding buffer for the nickel column.
    • 1L distilled H2O + 2.76g sodium phosphate + 29.22g NaCl + 1.36g imidazole
    • Final concentrations: 20mM sodium phosphate, 0.5M NaCl, 20mM imidazole
  3. Prepare a elution buffer for the nickel column.
    • 1L distilled H2O + 2.76g sodium phosphate + 29.22g NaCl + 34.05g imidazole
    • Final concentrations: 20mM sodium phosphate, 0.5M NaCl, 500mM imidazole
  4. Filter both of these solutions in a vacuum filter.

Prep for MBP-intein expression

  1. Combine 25g LB with 2g of D-glucose and 1L of distilled H2O in a 2800mL flask. (prepare 2)
  2. Combine 0.875g LB with 0.07g D-glucose and 35mL of distilled H2O in a 250mL flask. (prepare 2)
  3. Autoclave all of this media in a liquid cycle.