User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/02/29

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Objective

To analyze the data from the UV-Vis and fluorescence readings for GFP from yesterday. To prepare for expression of MBP-intein.

Description

The cultures from last night were spun down, resuspended, and transferred to the larger 1L LB cultures. Four hours later there was too much growth, and it was realized that the ampicillin was not added to the larger cultures, so they were now contaminated. The cultures were thrown out and a new expression will be completed next week.

Prep for MBP-intein expression

  1. Combine 25g LB with 2g of D-glucose and 1L of distilled H2O in a 2800mL flask. (prepare 4)
  2. Combine 0.875g LB with 0.07g D-glucose and 35mL of distilled H2O in a 250mL flask. (prepare 4)
  3. Autoclave all of this media in a liquid cycle.

Data Analysis from Yesterday

UV-Vis

Spectrum for GFP corrected with binding buffer spectrum:


Maximum absorbance is at 485nm. The corrected absorbance at this wavelength is 0.165.

A=εbc, where A485=0.165, b=1cm, and ε=61000cm-1M-1 The diluted concentration is calculated as 2.7μM. The concentration of the stock GFP is 27μM.

Fluorescence

Fluorescence of 10,000 times dilute GFP was taken. The protein was excited at 485nm, and an emmision scan was taken from 490nm - 700.5nm:




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