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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Live Cell Immunoassay (Eric))
Line 32: Line 32:
==Live Cell Immunoassay (Eric)==
==Live Cell Immunoassay (Eric)==
 +
*Today's assay for POLYSTYRENE
#Induce cells with current conditions
#Induce cells with current conditions
#Spin down 1 OD cells
#Spin down 1 OD cells

Revision as of 14:31, 30 July 2007

Mer-GFP Expression Protocol

  1. Mix Diluted LB Media (LB4.10)
    1. 4g NaCl, .5g Yeast Exract, 1g Tryptone + 1L H20
  2. Autoclave LB4.10
  3. Grow up 10 mls ON at 30°C
  4. Dilute back 1:100 in fresh LB4.10 (50 ul to 5 ml)
  5. Add HgCl2 in following concentrations (ng/ml):
    1. 0
    2. 5
    3. 10
    4. 25
    5. 50
    6. 75
    7. 100
    8. 150
    9. 300
    10. 500
    11. 1000
    12. 10000
  6. Grow at 30°C for 16h
  7. Wash a 3ml sample of each (twice) and resuspend in .9% NaCl (Minimizes Background)
  8. Measure flourescence
  • Excitation wavelength - 395nm
  • Emission wavelength - 509nm

==Measure out 0.1 g and dilute in 100 mL water This will give you 100 mL of 1 mg/mL Take 10 uL and dilute in 10 mL water This will give you 10 mL of 1 ug/mL Take 1 mL and dilute in 10 mL water This will give you 10 mL of 100 ng/mL

Live Cell Immunoassay (Eric)

  • Today's assay for POLYSTYRENE
  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
Personal tools