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Team:Washington/Protocols/VectorAssay
From 2010.igem.org
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------> | <!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------> | ||
| - | == GFP | + | == GFP EXPRESSION ASSAY == |
'''Day 1: OVERNIGHTS''' | '''Day 1: OVERNIGHTS''' | ||
| - | *Prepare a 96 deep well plate with 1ml of | + | *Prepare a 96 deep well plate with 1ml of LB + antibiotic |
*Inoculate well by taking at scraping from glycerol stock | *Inoculate well by taking at scraping from glycerol stock | ||
*Shake at 37deg 16-24hrs | *Shake at 37deg 16-24hrs | ||
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*Prepare 96 deep well plate with 1ml of TB + antibiotic, | *Prepare 96 deep well plate with 1ml of TB + antibiotic, | ||
| - | *Inoculate well by taking 20ul of the overnight and place it in | + | *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 | + | *Grow on shaker at 37C for 3 hours |
| - | *Induce all inducible constructs with | + | *Induce all inducible constructs with 50microL of 10mM IPTG |
*Allow to grow for 18 hour at room temperature on a shaker | *Allow to grow for 18 hour at room temperature on a shaker | ||
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*Take 100ul of PBS suspension and place into clear bottom plate reader plate | *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 | *Take 100ul of PBS suspension and place into black plate reader plate | ||
| - | *Read | + | *Read cell density by measuring absorbance at 600nm, and GFP fluorescence by exciting at 485 and reading emission at 525nm. |
Latest revision as of 04:50, 23 October 2010
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.
