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Team:Washington/Protocols/VectorAssay
From 2010.igem.org
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'''Day 1: OVERNIGHTS''' | '''Day 1: OVERNIGHTS''' | ||
Revision as of 03:35, 23 October 2010
GFP EXPRESSION ASSAY
Day 1: OVERNIGHTS
- Prepare a 96 deep well plate with 1ml of TB + 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 1 hour
- Induce all inducible constructs with 50ml 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 plates at 525nm and record data
