Team:Paris/July 31

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=== Results of the transformation ===
+
== Results of the transformation : Fluorescence ==
Line 8: Line 8:
|align="center"|'''Ligation name'''
|align="center"|'''Ligation name'''
|align="center"|'''Description'''
|align="center"|'''Description'''
-
|align="center"|'''Antibio'''
 
-
|align="center"|'''Nb colonies observed'''
 
|align="center"|'''Fluorescence'''
|align="center"|'''Fluorescence'''
|align="center"|'''Comments'''
|align="center"|'''Comments'''
-
|-
 
-
|align="center"|L100
 
-
|align="center"|
 
-
rbs TetR - ECFP
 
-
<br>D110 (BV) -  D130 (BI) 
 
-
|align="center"|
 
-
|align="center"|0
 
-
|align="center"|
 
-
|align="center"|To do again
 
-
|-
 
-
|align="center"|L101
 
-
|align="center"|
 
-
rbs TetR - GFP tripart
 
-
<br>D110 (BV) -  D131 (BI) 
 
-
|align="center"|
 
-
|align="center"|(1)
 
-
|align="center"|
 
-
|align="center"|To do again
 
|-
|-
|align="center"|L102
|align="center"|L102
-
|align="center"|
+
|align="center"|Strong rbs - YFP<br>D129 (BV) -  D118 (BI)   
-
Strong rbs - YFP
+
|align="center"|No
-
<br>D129 (BV) -  D118 (BI)   
+
|align="center"|OK
-
|align="center"|
+
-
|align="center"|+/- 100
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L103
|align="center"|L103
-
|align="center"|
+
|align="center"|Strong rbs - mRFP<br>D129 (BV) -  D122 (BI)   
-
Strong rbs - GFP
+
|align="center"|No
-
<br>D129 (BV) -  D122 (BI)   
+
|align="center"|OK
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L104
|align="center"|L104
-
|align="center"|
+
|align="center"|Strong rbs - lasR activator<br>D129 (BV) -  D114 (BI)   
-
Strong rbs - lasR activator
+
|align="center"|No
-
<br>D129 (BV) -  D114 (BI)   
+
|align="center"|OK
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L105
|align="center"|L105
-
|align="center"|
+
|align="center"|Strong promoter - ECFP<br>D123 (BV) - D130 (BI)
-
Strong promoter - ECFP
+
|align="center"|Yes (some others red because of the promoter)
-
<br>D123 (BV) - D130 (BI)
+
|align="center"|OK  
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
-
|align="center"|OK
+
|-
|-
|align="center"|L106
|align="center"|L106
-
|align="center"|
+
|align="center"|Strong promoter - gfp Tripart<br>D123 (BV) - D131 (BI)
-
Strong promoter - gfp Tripart
+
|align="center"|Yes (some others red <br> because of the promoter)
-
<br>D123 (BV) - D131 (BI)
+
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
|align="center"|OK
|align="center"|OK
|-
|-
|align="center"|L107
|align="center"|L107
-
|align="center"|
+
|align="center"|Strongest promoter - ECFP<br>D103 (BV) - D130 (BI)
-
Strongest promoter - ECFP
+
|align="center"|Yes (some others red <br>because of the promoter)
-
<br>D103 (BV) - D131 (BI)
+
|align="center"|OK
-
|align="center"|  
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L108 n°2 (the right one)
|align="center"|L108 n°2 (the right one)
-
|align="center"|
+
|align="center"|Strong promoter -  gfp Tripart<br>D103 (BV) - D131 (BI)
-
Strong promoter -  gfp Tripart
+
|align="center"|Yes (some others red because of the promoter)
-
<br>D103 (BV) - D130 (BI)
+
-
|align="center"|
+
-
|align="center"|+/- 100
+
-
|align="center"|
+
|align="center"|OK
|align="center"|OK
-
|-
 
-
|align="center"|L109
 
-
|align="center"|
 
-
Strong promoter -  ecfp
 
-
<br>D124 (BV) - D130 (BI)
 
-
|align="center"|
 
-
|align="center"|+/- 100
 
-
|align="center"|
 
-
|align="center"|To do again
 
|-
|-
|align="center"|L110
|align="center"|L110
-
|align="center"|
+
|align="center"|Medium promoter - gfp Tripart<br>D124 (BV) - D131 (BI)
-
Medium promoter - gfp Tripart
+
|align="center"|Yes (some others red because of the promoter)
-
<br>D124 (BV) - D131 (BI)
+
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
|align="center"|OK
|align="center"|OK
|-
|-
|align="center"|L111
|align="center"|L111
-
|align="center"|
+
|align="center"|Weak promoter - ECFP<br>D104 (BV) - D130 (BI)  
-
Weak promoter - ECFP
+
|align="center"|No
-
<br>D104 (BV) - D130 (BI)  
+
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
|align="center"|To do again
|align="center"|To do again
|-
|-
|align="center"|L112
|align="center"|L112
-
|align="center"|
+
|align="center"|Weak promoter - gfp tripart<br>D104 (BV) - D131 (BI)
-
Weak promoter - gfp
+
|align="center"|No
-
<br>D104 (BV) - D131 (BI)
+
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
-
|align="center"|To do again
+
-
|-
+
-
|align="center"|L113
+
-
|align="center"|
+
-
AracpBAD - ecfp
+
-
<br>D126 (BV) - D130 (BI)
+
-
|align="center"|
+
-
|align="center"|1
+
-
|align="center"|
+
-
|align="center"|To do again
+
-
|-
+
-
|align="center"|L114
+
-
|align="center"|
+
-
AracpBAD - gfp tripart
+
-
<br>D126 (BV) - D131 (BI)
+
-
|align="center"|
+
-
|align="center"|2
+
-
|align="center"|
+
|align="center"|To do again
|align="center"|To do again
|-
|-
|align="center"|L115
|align="center"|L115
-
|align="center"|
+
|align="center"|pLas - ECFP<br> D105 (BV)- D130 (BI)
-
pLas - ECFP
+
|align="center"|No
-
<br>D105 (BV) - D130 (BI)
+
|align="center"|OK
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L116
|align="center"|L116
-
|align="center"|
+
|align="center"|pLas - gfp Tripart<br>D105 (BV) - D131 (BI)
-
pLas - gfp Tripart
+
|align="center"|1 green
-
<br>D105 (BV) - D131 (BI)
+
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
|align="center"|To do again
|align="center"|To do again
|-
|-
|align="center"|L117
|align="center"|L117
-
|align="center"|
+
|align="center"|yfp - Double Terminator<br>D117 (FI) - D125 (FV)
-
yfp - Double Terminator
+
|align="center"|No
-
<br>D117 (FI) - D125 (FV)
+
|align="center"|OK
-
|align="center"|
+
-
|align="center"|+/- 30
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L118
|align="center"|L118
-
|align="center"|
+
|align="center"|rfp - Double Terminator<br>D121 (FI) - D125 (FV)
-
rfp - Double Terminator
+
|align="center"|No
-
<br>D121 (FI) - D125 (FV)
+
|align="center"|OK
-
|align="center"|
+
-
|align="center"|+/- 30
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L119
|align="center"|L119
-
|align="center"|
+
|align="center"|lasR activator + LVA - Double Terminator<br>D113 (FI) - D125 (FV)
-
lasR activator + LVA - Double Terminator
+
|align="center"|No
-
<br>D113 (FI) - D125 (FV)
+
|align="center"|OK
-
|align="center"|
+
-
|align="center"|+/- 30
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L120
|align="center"|L120
-
|align="center"|
+
|align="center"|tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI)
-
tetR repressible - ECFP
+
|align="center"|No
-
<br>D106 (BV) - D130 (BI)
+
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
|align="center"|To do again
|align="center"|To do again
|-
|-
|align="center"|L121
|align="center"|L121
-
|align="center"|
+
|align="center"|tetR repressible promoter - gfp tripart<br>D106 (BV) - D131 (BI)
-
Strong promoter - gfp tripart
+
|align="center"|Yes (some others red because of the promoter)
-
<br>D106 (BV) - D130 (BI)
+
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
|align="center"|OK
|align="center"|OK
-
|-
 
-
|align="center"|L122
 
-
|align="center"|
 
-
RBS-lasI - ecfp
 
-
<br>D107 (BV) - D130 (BI)
 
-
|align="center"|
 
-
|align="center"|0
 
-
|align="center"|
 
-
|align="center"|To do again
 
-
|-
 
-
|align="center"|L123
 
-
|align="center"|
 
-
RBS lasI - ecfp
 
-
<br>D107 (BV) - D131 (BI)
 
-
|align="center"|
 
-
|align="center"|1
 
-
|align="center"|
 
-
|align="center"|To do again
 
|-
|-
|align="center"|L124
|align="center"|L124
-
|align="center"|
+
|align="center"|Strongest RBS - mRFP<br>D102 (BV) - D122 (BI)
-
Strongest RBS - rfp
+
|align="center"|No
-
<br>D102 (BV) - D122 (BI)
+
|align="center"|OK
-
|align="center"|
+
-
|align="center"|a lot ++
+
-
|align="center"|
+
-
|align="center"|To do again
+
|-
|-
|align="center"|L125
|align="center"|L125
-
|align="center"|
+
|align="center"|Strongest RBS - YFP<br>D102 (BV) - D118 (BI)
-
Strongest RBS - yfp
+
|align="center"|No
-
<br>D102 (BV) - D118 (BI)
+
|align="center"|OK
-
|align="center"|
+
|}
-
|align="center"|a lot ++
+
 
-
|align="center"|
+
== Ligations ==
-
|align="center"|To do again
+
 
-
|-
+
''We tried again the following ligations :
-
|align="center"|L126
+
L100, L101, L109, L113, L114, L120, L122, L123, L126, L127''
-
|align="center"|
+
 
-
Strongest RBS (1)- LacR activator (+LVA)
+
=== Protocol ===
-
<br>D102 (BV) - D118 (BI)
+
 
-
|align="center"|
+
''For each samples,''  
-
|align="center"|no dish found!!!
+
 
-
|align="center"|
+
* 1 µl Ligase
-
|align="center"|To do again
+
* X µl Vector
-
|-
+
* Y µl Insert
-
|align="center"|L127
+
* 2 µl Ligase Buffer 10x
-
|align="center"|
+
* 20 µl qsp H2O
-
gfp (1)- Double terminator
+
 
-
<br>D119 (FI) - D125 (FV)
+
* Incubates O/N at 16°C (in the cold room).
-
|align="center"|
+
 
-
|align="center"|no dish found!!!
+
 
-
|align="center"|
+
=== List of new Ligation transformation ===
-
|align="center"|To do again
+
 
-
|-
+
{| border="1"  
-
|align="center"|'''Controls'''
+
|align=center|'''Name'''
-
|align="center"|
+
|align=center|'''Vector'''
-
|align="center"|
+
|align=center|'''Insert'''
-
|align="center"|
+
|align=center|'''Antibio'''
-
|align="center"|
+
|align=center|'''Vol. vector (µl)'''
-
|align="center"|
+
|align=center|'''Vol. insert (µl)'''
-
|-
+
|align=center|'''Vol. H20 (µl)'''
-
|align="center"|C1
+
-
|align="center"|
+
-
D110
+
-
|align="center"|
+
-
|align="center"|0
+
-
|align="center"|
+
-
|align="center"|-
+
-
|-
+
-
|align="center"|C2
+
-
|align="center"|
+
-
D129
+
-
|align="center"|
+
-
|align="center"|5
+
-
|align="center"|
+
-
|align="center"|-
+
-
|-
+
-
|align="center"|C3
+
-
|align="center"|
+
-
D123
+
-
|align="center"|
+
-
|align="center"|+/- 100
+
-
|align="center"|
+
-
|align="center"|-
+
|-
|-
-
|align="center"|C4
+
|style="background: #ccccff;"| L100
-
|align="center"|
+
|align=center| D110
-
D103
+
|align=center| D130
-
|align="center"|
+
|align=center| A
-
|align="center"|+/- 20
+
|align=center|3
-
|align="center"|
+
|align=center|6
-
|align="center"|-
+
|align=center|8
|-
|-
-
|align="center"|C5
+
|style="background: #ccccff;"| L101
-
|align="center"|
+
|align=center| D110
-
D124
+
|align=center| D131
-
|align="center"|
+
|align=center| A
-
|align="center"|+/- 100
+
|align=center|3
-
|align="center"|
+
|align=center|6
-
|align="center"|-
+
|align=center|8
|-
|-
-
|align="center"|C6
+
|style="background: #ccccff;"| L109
-
|align="center"|
+
|align=center| D124
-
D104
+
|align=center| D130
-
|align="center"|
+
|align=center| A
-
|align="center"|+/- 100
+
|align=center|4
-
|align="center"|
+
|align=center|7
-
|align="center"|-
+
|align=center|6
|-
|-
-
|align="center"|C7
+
|style="background: #ccccff;"| L113
-
|align="center"|
+
|align=center| D126
-
D126
+
|align=center| D130
-
|align="center"|
+
|align=center| K
-
|align="center"|0
+
|align=center|5
-
|align="center"|
+
|align=center|2.5
-
|align="center"|-
+
|align=center|9.5
|-
|-
-
|align="center"|C8
+
|style="background: #ccccff;"| L114
-
|align="center"|
+
|align=center| D126
-
D105
+
|align=center| D131
-
|align="center"|
+
|align=center| K
-
|align="center"|+/- 20
+
|align=center|5
-
|align="center"|
+
|align=center|2.5
-
|align="center"|-
+
|align=center|9.5
|-
|-
-
|align="center"|C9
+
|style="background: #ccccff;"| L120
-
|align="center"|
+
|align=center| D106
-
D125
+
|align=center| D130
-
|align="center"|
+
|align=center| A
-
|align="center"|0
+
|align=center|6
-
|align="center"|
+
|align=center|7.5
-
|align="center"|-
+
|align=center|3.5
|-
|-
-
|align="center"|C10
+
|style="background: #ccccff;"| L122
-
|align="center"|
+
|align=center| D107
-
D106
+
|align=center| D130
-
|align="center"|
+
|align=center| A
-
|align="center"|0
+
|align=center|7
-
|align="center"|
+
|align=center|3.5
-
|align="center"|-
+
|align=center|6.5
|-
|-
-
|align="center"|C11
+
|style="background: #ccccff;"| L123
-
|align="center"|
+
|align=center| D107
-
D107
+
|align=center| D131
-
|align="center"|
+
|align=center| A
-
|align="center"|0
+
|align=center|7
-
|align="center"|
+
|align=center|3.5
-
|align="center"|-
+
|align=center|6.5
|-
|-
-
|align="center"|C12
+
|style="background: #ccccff;"| L126
-
|align="center"|
+
|align=center| D102
-
D102
+
|align=center| D114
-
|align="center"|
+
|align=center| A
-
|align="center"|+/- 10
+
|align=center|6
-
|align="center"|
+
|align=center|4
-
|align="center"|-
+
|align=center|7
|-
|-
-
|align="center"|Positive control
+
|style="background: #ccccff;"| L127
-
|align="center"|
+
|align=center| D125
-
puc19
+
|align=center| D119
-
|align="center"|
+
|align=center| A
-
|align="center"|155 (transformation efficiency:1.5*10^7/ug)
+
|align=center|6
-
|align="center"|
+
|align=center|3
-
|align="center"|-
+
|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
Retrieved from "http://2008.igem.org/Team:Paris/July_31"