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Team:Washington/Protocols/VectorAssay
From 2010.igem.org
GFP EXPRESSION ASSAY
Day 1: OVERNIGHTS
- Prepare a 96 deep well plate with 1ml of LB + antibiotic
- Inoculate well by taking at scraping from glycerol stock
- Shake at 37deg 16-24hrs
Day 2: EXPRESSION
- Prepare 96 deep well plate with 1ml of TB + antibiotic,
- Inoculate well by taking 20ul of the overnight and place it in 1mL TB, do all inducible twice so to allow for induced verse uninducible expression
- Grow on shaker at 37C for 3 hours
- Induce all inducible constructs with 50microL of 10mM IPTG
- Allow to grow for 18 hour at room temperature on a shaker
Day 3: DATA
- Take overnights plates and spin on plate centrifuge for 20 minutes at 4000rpm
- Pour of broth and resuspend in 1ml PBS 7.5 pH on plate shaker
- Spin down PBS suspension for 20 minutes at 4000rpm
- Pour of supernatant
- Resuspend in 1ml PBS 7.5 pH on plate shaker
- Take 100ul of PBS suspension and place into clear bottom plate reader plate
- Take 100ul of PBS suspension and place into black plate reader plate
- Read cell density by measuring absorbance at 600nm, and GFP fluorescence by exciting at 485 and reading emission at 525nm.
