Team:Paris/July 31

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{{Paris/Calendar_Links|July 30|August 01}}
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{{Paris/Calendar_Links|July 30|August 1}}
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==Oligonucleotides design==
+
-
[[Image:RBS+.jpg|thumb|RBS +]]
 
-
[[Image:RBS-.jpg|thumb|RBS -]]
 
-
Creation of 3 oligonucleotides ([[team:Paris/Notebook/Oligo|O140, O141, O142]]) in order to create a particular plasmid
 
 +
== Results of the transformation : Fluorescence ==
-
 
-
==Transformation results==
 
{| border="1"  
{| border="1"  
|align="center"|'''Ligation name'''
|align="center"|'''Ligation name'''
-
|align="center"|'''Nb of colonies'''
 
|align="center"|'''Description'''
|align="center"|'''Description'''
-
|align="center"|'''Volume'''
+
|align="center"|'''Fluorescence'''
-
|-
+
|align="center"|'''Comments'''
-
|align="center"|L100
+
-
|align="center"|0
+
-
|align="center"|rbs-TetR <-> ECFP (RBS+LVA+Term)
+
-
|align="center"|3+6
+
-
|-
+
-
|align="center"|L101
+
-
|align="center"|(1)
+
-
|align="center"|rbs-TetR <-> GFP tripart+strong rbs
+
-
|align="center"|3+6
+
|-
|-
|align="center"|L102
|align="center"|L102
-
|align="center"|+/- 100
+
|align="center"|Strong rbs - YFP<br>D129 (BV) -  D118 (BI) 
-
|align="center"|strong RBS (0,6)<-> YFP
+
|align="center"|No
-
|align="center"|5+10
+
|align="center"|OK
|-
|-
|align="center"|L103
|align="center"|L103
-
|align="center"|a lot ++
+
|align="center"|Strong rbs - mRFP<br>D129 (BV) -  D122 (BI) 
-
|align="center"|strong RBS (0,6)<-> GFP
+
|align="center"|No
-
|align="center"|5+5
+
|align="center"|OK
|-
|-
|align="center"|L104
|align="center"|L104
-
|align="center"|a lot ++
+
|align="center"|Strong rbs - lasR activator<br>D129 (BV) -  D114 (BI) 
-
|align="center"|strong RBS (0,6)<-> LasR activator (+LVA)
+
|align="center"|No
-
|align="center"|5+4
+
|align="center"|OK
|-
|-
|align="center"|L105
|align="center"|L105
-
|align="center"|a lot ++
+
|align="center"|Strong promoter - ECFP<br>D123 (BV) - D130 (BI)
-
|align="center"|strong constitutive promoter <-> ECFP
+
|align="center"|Yes (some others red because of the promoter)
-
|align="center"|5+8
+
|align="center"|OK
|-
|-
|align="center"|L106
|align="center"|L106
-
|align="center"|a lot ++
+
|align="center"|Strong promoter - gfp Tripart<br>D123 (BV) - D131 (BI)
-
|align="center"|strong constitutive promoter <-> GFP tripart
+
|align="center"|Yes (some others red <br> because of the promoter)
-
|align="center"|5+8
+
|align="center"|OK
|-
|-
|align="center"|L107
|align="center"|L107
-
|align="center"|a lot ++
+
|align="center"|Strongest promoter - ECFP<br>D103 (BV) - D130 (BI)
-
|align="center"|strongest constitutive promoter <-> ECFP
+
|align="center"|Yes (some others red <br>because of the promoter)
-
|align="center"|5+8
+
|align="center"|OK
|-
|-
-
|align="center"|L108
+
|align="center"|L108 n°2 (the right one)
-
|align="center"|+/- 100 (2 dishes!?)
+
|align="center"|Strong promoter -  gfp Tripart<br>D103 (BV) - D131 (BI)
-
|align="center"|strongest constitutive promoter <-> GFP tripart
+
|align="center"|Yes (some others red because of the promoter)
-
|align="center"|5+8
+
|align="center"|OK
-
|-
+
-
|align="center"|L109
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-
|align="center"|+/- 30 or a lot ++ (2 dishes !?)  
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|align="center"|medium constitutive promoter <-> ECFP
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-
|align="center"|4+7
+
|-
|-
|align="center"|L110
|align="center"|L110
-
|align="center"|a lot ++
+
|align="center"|Medium promoter - gfp Tripart<br>D124 (BV) - D131 (BI)
-
|align="center"|medium constitutive promoter <-> gfp tripart
+
|align="center"|Yes (some others red because of the promoter)
-
|align="center"|4+7
+
|align="center"|OK
|-
|-
|align="center"|L111
|align="center"|L111
-
|align="center"|a lot ++
+
|align="center"|Weak promoter - ECFP<br>D104 (BV) - D130 (BI)
-
|align="center"|weak constitutive promoter <-> ECFP
+
|align="center"|No
-
|align="center"|3+5
+
|align="center"|To do again
|-
|-
|align="center"|L112
|align="center"|L112
-
|align="center"|a lot ++
+
|align="center"|Weak promoter - gfp tripart<br>D104 (BV) - D131 (BI)
-
|align="center"|weak constitutive promoter <-> gfp tripart
+
|align="center"|No
-
|align="center"|3+5
+
|align="center"|To do again
-
|-
+
-
|align="center"|L113
+
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|align="center"|1
+
-
|align="center"|Arac pBAD <-> ECFP
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-
|align="center"|5+2.5
+
-
|-
+
-
|align="center"|L114
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-
|align="center"|2
+
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|align="center"|Arac pBAD <-> gfp tripart
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-
|align="center"|5+2.5
+
|-
|-
|align="center"|L115
|align="center"|L115
-
|align="center"|a lot ++
+
|align="center"|pLas - ECFP<br> D105 (BV)- D130 (BI)
-
|align="center"|pLas <-> ECFP
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|align="center"|No
-
|align="center"|7+11
+
|align="center"|OK
|-
|-
-
|align="center"|L110
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|align="center"|L116
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|align="center"|a lot ++
+
|align="center"|pLas - gfp Tripart<br>D105 (BV) - D131 (BI)
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|align="center"|pLas <-> gfp tripart
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|align="center"|1 green
-
|align="center"|7+11
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|align="center"|To do again
|-
|-
|align="center"|L117
|align="center"|L117
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|align="center"|C0040
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|align="center"|yfp - Double Terminator<br>D117 (FI) - D125 (FV)
-
|align="center"|A
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|align="center"|No
-
|align="center"|0 CFU
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|align="center"|OK
 +
|-
 +
|align="center"|L118
 +
|align="center"|rfp - Double Terminator<br>D121 (FI) - D125 (FV)
 +
|align="center"|No
 +
|align="center"|OK
 +
|-
 +
|align="center"|L119
 +
|align="center"|lasR activator + LVA - Double Terminator<br>D113 (FI) - D125 (FV)
 +
|align="center"|No
 +
|align="center"|OK
 +
|-
 +
|align="center"|L120
 +
|align="center"|tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI)
 +
|align="center"|No
 +
|align="center"|To do again
 +
|-
 +
|align="center"|L121
 +
|align="center"|tetR repressible promoter - gfp tripart<br>D106 (BV) - D131 (BI)
 +
|align="center"|Yes (some others red because of the promoter)
 +
|align="center"|OK
 +
|-
 +
|align="center"|L124
 +
|align="center"|Strongest RBS - mRFP<br>D102 (BV) - D122 (BI)
 +
|align="center"|No
 +
|align="center"|OK
 +
|-
 +
|align="center"|L125
 +
|align="center"|Strongest RBS - YFP<br>D102 (BV) - D118 (BI)
 +
|align="center"|No
 +
|align="center"|OK
 +
|}
 +
 
 +
== Ligations ==
 +
 
 +
''We tried again the following ligations :
 +
L100, L101, L109, L113, L114, L120, L122, L123, L126, L127''
 +
 
 +
=== Protocol ===
 +
 
 +
''For each samples,''
 +
 
 +
* 1 µl Ligase
 +
* X µl Vector
 +
* Y µl Insert
 +
* 2 µl Ligase Buffer 10x
 +
* 20 µl qsp H2O
 +
 
 +
* Incubates O/N at 16°C (in the cold room).
 +
 
 +
 
 +
=== List of new Ligation transformation ===
 +
 
 +
{| border="1"
 +
|align=center|'''Name'''
 +
|align=center|'''Vector'''
 +
|align=center|'''Insert'''
 +
|align=center|'''Antibio'''
 +
|align=center|'''Vol. vector (µl)'''
 +
|align=center|'''Vol. insert (µl)'''
 +
|align=center|'''Vol. H20 (µl)'''
 +
|-
 +
|style="background: #ccccff;"| L100
 +
|align=center| D110
 +
|align=center| D130
 +
|align=center| A
 +
|align=center|3
 +
|align=center|6
 +
|align=center|8
 +
|-
 +
|style="background: #ccccff;"| L101
 +
|align=center| D110
 +
|align=center| D131
 +
|align=center| A
 +
|align=center|3
 +
|align=center|6
 +
|align=center|8
 +
|-
 +
|style="background: #ccccff;"|  L109
 +
|align=center| D124
 +
|align=center| D130
 +
|align=center| A
 +
|align=center|4
 +
|align=center|7
 +
|align=center|6
 +
|-
 +
|style="background: #ccccff;"|  L113
 +
|align=center| D126
 +
|align=center| D130
 +
|align=center| K
 +
|align=center|5
 +
|align=center|2.5
 +
|align=center|9.5
 +
|-
 +
|style="background: #ccccff;"|  L114
 +
|align=center| D126
 +
|align=center| D131
 +
|align=center| K
 +
|align=center|5
 +
|align=center|2.5
 +
|align=center|9.5
 +
|-
 +
|style="background: #ccccff;"|  L120
 +
|align=center| D106
 +
|align=center| D130
 +
|align=center| A
 +
|align=center|6
 +
|align=center|7.5
 +
|align=center|3.5
|-
|-
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|align="center"|1C_SP 1008
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|style="background: #ccccff;"| L122
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|align="center"|PSB1A3
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|align=center| D107
-
|align="center"|A
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|align=center| D130
-
|align="center"|few
+
|align=center| A
 +
|align=center|7
 +
|align=center|3.5
 +
|align=center|6.5
|-
|-
-
|align="center"|7G_SP 1013
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|style="background: #ccccff;"| L123
-
|align="center"|E1010
+
|align=center| D107
-
|align="center"|K
+
|align=center| D131
-
|align="center"|medium
+
|align=center| A
 +
|align=center|7
 +
|align=center|3.5
 +
|align=center|6.5
|-
|-
-
|align="center"|7E_SP 1001
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|style="background: #ccccff;"| L126
-
|align="center"|E0040
+
|align=center| D102
-
|align="center"|A
+
|align=center| D114
-
|align="center"|2 CFU
+
|align=center| A
 +
|align=center|6
 +
|align=center|4
 +
|align=center|7
|-
|-
-
|align="center"|2F_SP 1016
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|style="background: #ccccff;"| L127
-
|align="center"|E0030
+
|align=center| D125
-
|align="center"|K
+
|align=center| D119
-
|align="center"|a lot
+
|align=center| A
 +
|align=center|6
 +
|align=center|3
 +
|align=center|8
|}
|}

Latest revision as of 17:04, 13 August 2008

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Contents

Results of the transformation : Fluorescence

Ligation name Description Fluorescence Comments
L102 Strong rbs - YFP
D129 (BV) - D118 (BI) 		No OK
L103 Strong rbs - mRFP
D129 (BV) - D122 (BI) 		No OK
L104 Strong rbs - lasR activator
D129 (BV) - D114 (BI) 		No OK
L105 Strong promoter - ECFP
D123 (BV) - D130 (BI) 		Yes (some others red because of the promoter) OK
L106 Strong promoter - gfp Tripart
D123 (BV) - D131 (BI) 		Yes (some others red
because of the promoter) 		OK
L107 Strongest promoter - ECFP
D103 (BV) - D130 (BI) 		Yes (some others red
because of the promoter) 		OK
L108 n°2 (the right one) Strong promoter - gfp Tripart
D103 (BV) - D131 (BI) 		Yes (some others red because of the promoter) OK
L110 Medium promoter - gfp Tripart
D124 (BV) - D131 (BI) 		Yes (some others red because of the promoter) OK
L111 Weak promoter - ECFP
D104 (BV) - D130 (BI) 		No To do again
L112 Weak promoter - gfp tripart
D104 (BV) - D131 (BI) 		No To do again
L115 pLas - ECFP
D105 (BV)- D130 (BI) 		No OK
L116 pLas - gfp Tripart
D105 (BV) - D131 (BI) 		1 green To do again
L117 yfp - Double Terminator
D117 (FI) - D125 (FV) 		No OK
L118 rfp - Double Terminator
D121 (FI) - D125 (FV) 		No OK
L119 lasR activator + LVA - Double Terminator
D113 (FI) - D125 (FV) 		No OK
L120 tetR repressible promoter - ECFP
D106 (BV) - D130 (BI) 		No To do again
L121 tetR repressible promoter - gfp tripart
D106 (BV) - D131 (BI) 		Yes (some others red because of the promoter) OK
L124 Strongest RBS - mRFP
D102 (BV) - D122 (BI) 		No OK
L125 Strongest RBS - YFP
D102 (BV) - D118 (BI) 		No OK

Ligations

We tried again the following ligations : L100, L101, L109, L113, L114, L120, L122, L123, L126, L127

Protocol

For each samples,

  • 1 µl Ligase
  • X µl Vector
  • Y µl Insert
  • 2 µl Ligase Buffer 10x
  • 20 µl qsp H2O
  • Incubates O/N at 16°C (in the cold room).


List of new Ligation transformation

Name Vector Insert Antibio Vol. vector (µl) Vol. insert (µl) Vol. H20 (µl)
L100 D110 D130 A 3 6 8
L101 D110 D131 A 3 6 8
L109 D124 D130 A 4 7 6
L113 D126 D130 K 5 2.5 9.5
L114 D126 D131 K 5 2.5 9.5
L120 D106 D130 A 6 7.5 3.5
L122 D107 D130 A 7 3.5 6.5
L123 D107 D131 A 7 3.5 6.5
L126 D102 D114 A 6 4 7
L127 D125 D119 A 6 3 8
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