Team:Washington/Protocols/VectorAssay

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{{Template:Team:Washington/Templates/Header}}
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS---------------------------------------->
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== GFP EXPRESSION ASSAY ==
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'''Day 1:  OVERNIGHTS'''
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*Prepare a 96 deep well plate with 1ml of LB + antibiotic
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*Inoculate well by taking at scraping from glycerol stock
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*Shake at 37deg 16-24hrs
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'''Day 2: EXPRESSION'''
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*Prepare 96 deep well plate with 1ml of TB + antibiotic,
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*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
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*Grow on shaker at 37C for 3 hours
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*Induce all inducible constructs with 50microL of 10mM IPTG
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*Allow to grow for 18 hour at room temperature on a shaker
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'''Day 3: DATA'''
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*Take overnights plates and spin on plate centrifuge for 20 minutes at 4000rpm
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*Pour of broth and resuspend in 1ml PBS 7.5 pH on plate shaker
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*Spin down PBS suspension for 20 minutes at 4000rpm
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*Pour of supernatant
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*Resuspend in 1ml PBS 7.5 pH on plate shaker
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*Take 100ul of PBS suspension and place into clear bottom plate reader plate
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*Take 100ul of PBS suspension and place into black plate reader plate
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*Read cell density by measuring absorbance at 600nm, and GFP fluorescence by exciting at 485 and reading emission at 525nm.
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<!---------------------------------------PAGE CONTENT GOES ABOVE THIS---------------------------------------->
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<div style="text-align:center">
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'''&larr; [[Team:Washington/Protocols|Back to Lab Protocols]]'''
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&nbsp; &nbsp; &nbsp;
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</div>
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{{Template:Team:Washington/Templates/Footer}}

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.


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