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==
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Creation of 3 oligonucleotides ([[team:Paris/Notebook/Oligo|O140, O141, O142]])in order to create a particular plasmid
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[[Image:RBS+.jpg|thumb|RBS +]]
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== Results of the transformation : Fluorescence ==
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[[Image:RBS-.jpg|thumb|RBS -]]
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 +
 
 +
{| border="1"
 +
|align="center"|'''Ligation name'''
 +
|align="center"|'''Description'''
 +
|align="center"|'''Fluorescence'''
 +
|align="center"|'''Comments'''
 +
|-
 +
|align="center"|L102
 +
|align="center"|Strong rbs - YFP<br>D129 (BV) -  D118 (BI) 
 +
|align="center"|No
 +
|align="center"|OK
 +
|-
 +
|align="center"|L103
 +
|align="center"|Strong rbs - mRFP<br>D129 (BV) -  D122 (BI) 
 +
|align="center"|No
 +
|align="center"|OK
 +
|-
 +
|align="center"|L104
 +
|align="center"|Strong rbs - lasR activator<br>D129 (BV) -  D114 (BI) 
 +
|align="center"|No
 +
|align="center"|OK
 +
|-
 +
|align="center"|L105
 +
|align="center"|Strong promoter - ECFP<br>D123 (BV) - D130 (BI)
 +
|align="center"|Yes (some others red because of the promoter)
 +
|align="center"|OK
 +
|-
 +
|align="center"|L106
 +
|align="center"|Strong promoter - gfp Tripart<br>D123 (BV) - D131 (BI)
 +
|align="center"|Yes (some others red <br> because of the promoter)
 +
|align="center"|OK
 +
|-
 +
|align="center"|L107
 +
|align="center"|Strongest promoter - ECFP<br>D103 (BV) - D130 (BI)
 +
|align="center"|Yes (some others red <br>because of the promoter)
 +
|align="center"|OK
 +
|-
 +
|align="center"|L108 n°2 (the right one)
 +
|align="center"|Strong promoter -  gfp Tripart<br>D103 (BV) - D131 (BI)
 +
|align="center"|Yes (some others red because of the promoter)
 +
|align="center"|OK
 +
|-
 +
|align="center"|L110
 +
|align="center"|Medium promoter - gfp Tripart<br>D124 (BV) - D131 (BI)
 +
|align="center"|Yes (some others red because of the promoter)
 +
|align="center"|OK
 +
|-
 +
|align="center"|L111
 +
|align="center"|Weak promoter - ECFP<br>D104 (BV) - D130 (BI)
 +
|align="center"|No
 +
|align="center"|To do again
 +
|-
 +
|align="center"|L112
 +
|align="center"|Weak promoter - gfp tripart<br>D104 (BV) - D131 (BI)
 +
|align="center"|No
 +
|align="center"|To do again
 +
|-
 +
|align="center"|L115
 +
|align="center"|pLas - ECFP<br> D105 (BV)- D130 (BI)
 +
|align="center"|No
 +
|align="center"|OK
 +
|-
 +
|align="center"|L116
 +
|align="center"|pLas - gfp Tripart<br>D105 (BV) - D131 (BI)
 +
|align="center"|1 green
 +
|align="center"|To do again
 +
|-
 +
|align="center"|L117
 +
|align="center"|yfp - Double Terminator<br>D117 (FI) - D125 (FV)
 +
|align="center"|No
 +
|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
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|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 :
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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'''
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|align=center|'''Vector'''
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|align=center|'''Insert'''
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|align=center|'''Antibio'''
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|align=center|'''Vol. vector (µl)'''
 +
|align=center|'''Vol. insert (µl)'''
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|align=center|'''Vol. H20 (µl)'''
 +
|-
 +
|style="background: #ccccff;"| L100
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|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
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|align=center| D131
 +
|align=center| K
 +
|align=center|5
 +
|align=center|2.5
 +
|align=center|9.5
 +
|-
 +
|style="background: #ccccff;"|  L120
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|align=center| D106
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|align=center| D130
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|align=center| A
 +
|align=center|6
 +
|align=center|7.5
 +
|align=center|3.5
 +
|-
 +
|style="background: #ccccff;"|  L122
 +
|align=center| D107
 +
|align=center| D130
 +
|align=center| A
 +
|align=center|7
 +
|align=center|3.5
 +
|align=center|6.5
 +
|-
 +
|style="background: #ccccff;"| L123
 +
|align=center| D107
 +
|align=center| D131
 +
|align=center| A
 +
|align=center|7
 +
|align=center|3.5
 +
|align=center|6.5
 +
|-
 +
|style="background: #ccccff;"|  L126
 +
|align=center| D102
 +
|align=center| D114
 +
|align=center| A
 +
|align=center|6
 +
|align=center|4
 +
|align=center|7
 +
|-
 +
|style="background: #ccccff;"|  L127
 +
|align=center| D125
 +
|align=center| D119
 +
|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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