Team:Washington/Protocols/VectorAssay

From 2010.igem.org

(Difference between revisions)
(GFP expression assay)
 
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'''Day 1:  OVERNIGHTS'''
'''Day 1:  OVERNIGHTS'''
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*Prepare a 96 deep well plate with 1ml of TB + antibiotic
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*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 1ml TB, do all inducible twice so to allow for induced verse uninducible expression
+
*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
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*Grow on shaker at 37C for 3 hours
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*Induce all inducible constructs with 50ml of 10mM IPTG
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*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
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*Read plates at 525nm and record data
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*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.


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