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
- Add 35μL of ampicillin to each of the 250mL flasks (previously autoclaved with 35mL of LB with glucose).
- 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.
- Incubate the flasks overnight at 250rpm at 37°C.
UV-Vis Spectrum of GFP
- 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.
- Take a spectrum from 200nm-800nm of the solution.
- Also, take a spectrum from 200nm-800nm of 1mL of the binding buffer.
- Determine the wavelength with the maximum absorbance using the GFP spectrum corrected for the binding buffer.
Fluorescence of GFP
- Dilute the GFP 10,000 times (serial dilutions with binding buffer). Put 3mL in a quartz cuvette.
- Excite the solution at 485nm, and scan from 490nm - 700.5nm.
Data
Data will be analyzed tomorrow.
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