IGEM:MIT/2007/Notebook/2007-7-30

Mer-GFP Expression Protocol

 * 1) Mix Diluted LB Media (LB4.10)
 * 2) 4g NaCl, .5g Yeast Exract, 1g Tryptone + 1L H20
 * 3) Autoclave LB4.10
 * 4) Grow up 10 mls ON at 30°C
 * 5) Dilute back 1:100 in fresh LB4.10 (50 ul to 5 ml)
 * 6) Add HgCl2 in following concentrations (ng/ml):
 * 0
 * 5
 * 10
 * 25
 * 50
 * 75
 * 1) 100
 * 2) 150
 * 3) 300
 * 4) 500
 * 5) 1000
 * 6) 10000
 * 7) Grow at 30°C for 16h
 * 8) Wash a 3ml sample of each (twice) and resuspend in .9% NaCl (Minimizes Background)
 * 9) 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
 * 1) Induce cells with current conditions
 * 2) Spin down 1 OD cells
 * 3) Resuspend in 1 mL PBS-BSA (5 mg/mL) -- make 50 mL
 * 4) Spin down 0.2 OD (200 µl); aspirate off media
 * 5) Resuspend in 100 µl 1:500 (experiment?) antiT7 antibody in PBS-BSA (1 mL PBS-BSA; 2 µl antiT7)
 * 6) Incubate on ice for 30 minutes
 * 7) Add 800 µl cold PBS-BSA
 * 8) Spin down cells and remove media
 * 9) Resuspend in 100 µl 1:20 anti-mouse antibody in PBS-BSA (1 mL PBS-BSA; 50 µl anti-mouse)
 * 10) Incubate on ice for 30 minutes IN THE DARK
 * 11) Repeat Step 7
 * 12) Repeat Step 8
 * 13) Repeat Steps 7 and 8; wash
 * 14) Resuspend in 100 µl PBS-BSA
 * 15) Look under fluorescent microscope
 * Do induced + controls