IGEM:MIT/2007/Notebook/2007-7-30
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Mer-GFP Expression Protocol
- Mix Diluted LB Media (LB4.10)
- 4g NaCl, .5g Yeast Exract, 1g Tryptone + 1L H20
- Autoclave LB4.10
- Grow up 10 mls ON at 30°C
- Dilute back 1:100 in fresh LB4.10 (50 ul to 5 ml)
- Add HgCl2 in following concentrations (ng/ml):
- 0
- 5
- 10
- 25
- 50
- 75
- 100
- 150
- 300
- 500
- 1000
- 10000
- Grow at 30°C for 16h
- Wash a 3ml sample of each (twice) and resuspend in .9% NaCl (Minimizes Background)
- Measure flourescence
- Excitation wavelength - 395nm
- Emission wavelength - 509nm
Measure out 0.1 g and dilute in 10 mL water This will give you 10 mL of 10 mg/mL Take 100 uL and dilute in 10 mL water This will give you 10 mL of 100 ug/mL Take 100 ul and dilute in 10 mL water This will give you 10 mL of 1 ug/mL
Live Cell Immunoassay (Eric)
- Today's assay - fluorescent staining of T7 tag engineered into the CPX insert. Checks for expression of CPX and polystyrene-binding peptide
- Induce cells with current conditions
- Spin down 1 OD cells
- Resuspend in 1 mL PBS-BSA (5 mg/mL) -- make 50 mL
- Spin down 0.2 OD (200 µl); aspirate off media
- Resuspend in 100 µl 1:500 (experiment?) antiT7 antibody in PBS-BSA (1 mL PBS-BSA; 2 µl antiT7)
- Incubate on ice for 30 minutes
- Add 800 µl cold PBS-BSA
- Spin down cells and remove media
- Resuspend in 100 µl 1:20 anti-mouse antibody in PBS-BSA (1 mL PBS-BSA; 50 µl anti-mouse)
- Incubate on ice for 30 minutes IN THE DARK
- Repeat Step 7
- Repeat Step 8
- Repeat Steps 7 and 8; wash
- Resuspend in 100 µl PBS-BSA
- Look under fluorescent microscope
- Do induced + controls
Results of Immuno-Stainng Assay
-
AHL-6 1
-
AHL-6 2
-
AHL-6 3
-
AHL-7 1
-
AHL-7 2
-
AHL-7 3
-
AHL-8 1
-
AHL-8 2
-
AHL-8 3
-
Control 1
-
Control 2
-
Control 3