Talk:Tokyo/Preliminary assays

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== [[Tokyo/AHL preparation |AHL preparation]] ==
 
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== [[Tokyo/Experimental precision and accurecy|Experimental precision and accurecy]] ==
 
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== AHL assay==
 
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===Purpose1: ===
 
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To practice AHL assay.
 
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===Purpose2: ===
 
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To check if AHL works. 
 
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===Samples: ===
 
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<br>luxR + GFP in DH5a (AHL-activated promoter)
 
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<br>placQI + GFP in DH5a (pos. con.)
 
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<br>pSBΔP+ GFP (neg. con.)
 
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<br>ΔP = promoter deleted
 
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===Procedure: ===
 
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<br>The final concentration of AHL is 6 pM.
 
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===Result: ===
 
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===Conclusion: ===
 
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6pM of AHL is not enough to activate the AHL-activated luxR promoter.
 
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== AHL assay==
 
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===Purpose: ===
 
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check if lux-lac hybrid promoter works
 
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===Samples: ===
 
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<br>Lux-lac hybrid promoter (before blue light check)
 
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<br>Lux-lac hybrid promoter (after blue light check)
 
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<br>placQI (pos. con.)
 
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<br>modified A4 (promoter deleted) (neg. con.)
 
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===Procedure: ===
 
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[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
 
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===Result: ===
 
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===Conclusion: ===
 
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== OD correction ==
 
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===Purpose: ===
 
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<br>To see how the fluorescence changes though time course.
 
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===Samples: ===
 
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<br>ΔP-GFP in pTrc99A (neg. con.)
 
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<br>placQI in pTrc99A (pos. con.)
 
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<br>Only LB (blank)
 
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===Procedure: ===
 
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[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
 
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===Result: ===
 
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===Conclusion: ===
 
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== Wash ==
 
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===Purpose: ===
 
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To determine the actual fluorescence of GFP expressed under the promoters by removing other influencial facters such as culture media.
 
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===Samples: ===
 
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<br>A4 placQI in pTrc99A (pos. con.)
 
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<br>A4ΔP in pTrc99A
 
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<br>A4 hybrid promoter in pBR322
 
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===Procedure: ===
 
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[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
 
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<br>[[Tokyo/Wash |Wash ]]
 
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===Result: ===
 
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===Conclusion: ===
 
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== Detailed AHL assay ==
 
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===Purpose: ===
 
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<br>To determine the AHL dependent behaviour in narrower range of AHL concentration for the analyses and simulation.
 
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<br>To deternime the Hill coefficient for the relation between the concentration of AHL and our lux^lac hybrid promoter.
 
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===Samples: ===
 
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<br>A4 placQI in pTrc99A (pos. con.)
 
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<br>A4ΔP in pTrc99A (neg. con.)
 
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<br>A4 hybrid promoter in pBR322
 
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===Procedure: ===
 
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<br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
 
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<br>[[Tokyo/Wash |Wash ]]
 
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===Result: ===
 
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===Conclusion: ===
 
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== New-AHL assay ==
 
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===Purpose: ===
 
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<br>To determine and compare the activity of the AHL used so far and that of the newly perchased AHL.
 
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===Samples: ===
 
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<br>A4 placQI in pTrc99A (pos. con.)
 
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<br>A4ΔP in pTrc99A (neg. con.)
 
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<br>A4 hybrid promoter in pBR322
 
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===Procedure: ===
 
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<br>The old and the newly delivered AHL was used.
 
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<br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
 
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<br>The final concantrations of AHL applied here were 0.01, 0.1, 0.5, 1, 10, 100, and 1000 nM.
 
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===Result: ===
 
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===Conclusion: ===
 
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== Lux-lac hybrid promoter Check ==
 
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[[Image:luxlachybrid.jpg]]
 
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===Purpose 1:===
 
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To check if LacI hybrid promoter is activated by AHL and repressed by LacI.
 
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===Purpose 2:===
 
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To obtain parameters of LacI hybrid promoter for computational simulation.
 
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===Samples: ===
 
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<br>1. pTrc99A + [LacI hybrid promoter – GFP] (LacI+)
 
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<br>2. pBR322 TetR + [LacI hybrid promoter – GFP] (lacI-)
 
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<br>3. pTrc99A + [placQI – GFP] (placQI is constitutive promoter)
 
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<br>4. pTrc99A + [LacI promoter – GFP]
 
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<br>5. pBR322 TetR + [LacI promoter – GFP]
 
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<br>'''Repressor LacI expression:'''
 
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<br>pTrc99A expresses LacI
 
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<br>pBR322 TetR does NOT express LacI
 
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<br>'''Antibiotics resistance:'''
 
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<br>pTrc99A gives ampicillin-resistance
 
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<br>pBR322 TetR gives ampicillin-resistance
 
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<br>[LacI hybrid promoter – GFP] gives kanamicin-resistance
 
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<br>[placQI – GFP] gives kanamicin-resistance
 
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===Procedure:===
 
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<br>[[Tokyo/AHL assay Standard protocol |AHL assay Standard protocol ]]
 

Revision as of 03:15, 22 October 2007