User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/02/28

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Objective

Finish preparing for, and begin the expression of MBP-intein in E.coli. Take UV-Vis spectra and fluorescence of GFP.

Description

Preparation for Expression

  • Prepare 5mL 0.1 M Isopropyl β-D-1-thiogalactopyranoside (IPTG) solution
    • 0.12g + 5mL distilled H2O
    • Sterile Filter the solution
  • Prepare 5mL 100mg/mL Ampicillin
    • 0.5g ampicillin + 5mL distilled H2O
    • Sterile Filter the solution

Begin Expression of MBP-intein

  1. Add 35μL of ampicillin to each of the 250mL flasks (previously autoclaved with 35mL of LB with glucose).
  2. Add one colony from the previously streaked out LB/Amp plate (02/01/12 from glycerol stock pMXB10 Stock #115) to each of these flasks.
  3. Incubate the flasks overnight at 250rpm at 37°C.

UV-Vis Spectrum of GFP

  1. Dilute the GFP ten times, by mixing 100μL of the GFP with 900μL of the binding buffer (20mM sodium phosphate, 0.5M NaCl, 20mM imidazole) in a 1mL quartz cuvette.
  2. Take a spectrum from 200nm-800nm of the solution.
  3. Also, take a spectrum from 200nm-800nm of 1mL of the binding buffer.
  4. Determine the wavelength with the maximum absorbance using the GFP spectrum corrected for the binding buffer.

Fluorescence of GFP

  1. Dilute the GFP 10,000 times (serial dilutions with binding buffer). Put 3mL in a quartz cuvette.
  2. Excite the solution at 485nm, and scan from 490nm - 700.5nm.

Data

Data will be analyzed tomorrow.



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