Team:Paris/July 26

From 2008.igem.org

(Difference between revisions)
(→Extraction of the DNA)
 
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=== Results of digestions : Electrophoresis ===
=== Results of digestions : Electrophoresis ===
-
Gel 1, 2, 3, 4, 5 = '''0.8%'''    et    Gel 6 = '''1,5%'''
+
'''conditions :'''
 +
 +
* 10µl of ladder 1 kb (except for gel n°6 : 100 pb)
 +
* 30µl of digestion added with 5µl of loading Dye 6x
 +
* migration ~30min at 100W
 +
* Gel 1, 2, 3, 4, 5 = '''0.8%'''     
 +
* Gel 6 = '''1,5%'''
Line 125: Line 131:
-
gel5 [[Image:‎080726gel_5.jpg| gel5|100px]]
+
gel5 [[Image:080726gel_5.jpg| gel5|100px]]
-
gel6 [[Image:‎080726gel_6.jpg| gel6|100px]]
+
gel6 [[Image:080726gel_6.jpg| gel6|100px]]
-
{| border="1"  
+
{|- border="1"
-
|align=center|'''Name'''
+
|align=center style="background: #649CD7;"|'''Name'''
-
|align=center|'''BioBrick'''
+
|align=center style="background: #649CD7;"|'''BioBrick'''
-
|align=center|'''Tube N°'''
+
|align=center style="background: #649CD7;"|'''Tube N°'''
-
|align=center|'''Enz 1'''
+
|align=center style="background: #649CD7;"|'''Enz 1'''
-
|align=center|'''Enz 2'''
+
|align=center style="background: #649CD7;"|'''Enz 2'''
-
|align=center|'''Obs'''
+
|align=center style="background: #649CD7;"|'''Obs'''
-
|align=center|'''Exp Size Matrix'''
+
|align=center style="background: #649CD7;"|'''Exp Size Matrix'''
-
|align=center|'''Exp Size BB'''
+
|align=center style="background: #649CD7;"|'''Exp Size BB'''
-
|align=center|'''Mea Size Matrix'''
+
|align=center style="background: #649CD7;"|'''Mea Size Matrix'''
-
|align=center|'''Mea Size BB'''
+
|align=center style="background: #649CD7;"|'''Mea Size BB'''
-
|align=center|'''Gel'''
+
|align=center style="background: #649CD7;"|'''Gel'''
-
|align=center|'''Band'''
+
|align=center style="background: #649CD7;"|'''Band'''
|-
|-
-
! rowspan="5" style="background: #ccccff;" | D100
+
! rowspan="2" style="background: #D4E2EF;" | D100
-
! rowspan="5" style="background: #ccccff;" | B0034
+
! rowspan="2" style="background: #D4E2EF;" | B0034
|align=center|1
|align=center|1
! rowspan="2"| XbaI
! rowspan="2"| XbaI
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! rowspan="1"| 3
! rowspan="1"| 3
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D101
 +
! rowspan="1" style="background: #D4E2EF;" | B0034
|align=center|3
|align=center|3
|align=center| EcoRI
|align=center| EcoRI
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|align=center| 2
|align=center| 2
|-
|-
 +
! rowspan="2" style="background: #D4E2EF;" | D102
 +
! rowspan="2" style="background: #D4E2EF;" | B0034
|align=center|4
|align=center|4
! rowspan="2"| SpeI
! rowspan="2"| SpeI
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|align=center| 4
|align=center| 4
|-
|-
-
! rowspan="2" style="background: #ccccff;" | D101
+
! rowspan="2" style="background: #D4E2EF;" | D103
-
! rowspan="2" style="background: #ccccff;" | J23101
+
! rowspan="2" style="background: #D4E2EF;" | J23101
|align=center|1
|align=center|1
! rowspan="2"| SpeI
! rowspan="2"| SpeI
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|align=center| 6
|align=center| 6
|-
|-
-
! rowspan="2" style="background: #ccccff;" | D102
+
! rowspan="2" style="background: #D4E2EF;" | D104
-
! rowspan="2" style="background: #ccccff;" | J23109
+
! rowspan="2" style="background: #D4E2EF;" | J23109
|align=center|1
|align=center|1
! rowspan="2"| SpeI
! rowspan="2"| SpeI
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! rowspan="1"| 8
! rowspan="1"| 8
|-
|-
-
! rowspan="2" style="background: #ccccff;" | D103
+
! rowspan="2" style="background: #D4E2EF;" | D105
-
! rowspan="2" style="background: #ccccff;" | R0079
+
! rowspan="2" style="background: #D4E2EF;" | R0079
|align=center|1
|align=center|1
! rowspan="2"| SpeI
! rowspan="2"| SpeI
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|align=center| 10
|align=center| 10
|-
|-
-
! rowspan="2" style="background: #ccccff;" | D104
+
! rowspan="2" style="background: #D4E2EF;" | D106
-
! rowspan="2" style="background: #ccccff;" | R0040
+
! rowspan="2" style="background: #D4E2EF;" | R0040
|align=center|1
|align=center|1
! rowspan="2"| SpeI
! rowspan="2"| SpeI
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|align=center| 12
|align=center| 12
|-
|-
-
! rowspan="4" style="background: #ccccff;" | D105
+
! rowspan="1" style="background: #D4E2EF;" | D107
-
! rowspan="4" style="background: #ccccff;" | S03154
+
! rowspan="1" style="background: #D4E2EF;" | S03154
|align=center|1
|align=center|1
! rowspan="1"| SpeI
! rowspan="1"| SpeI
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|align=center| 5
|align=center| 5
|-
|-
 +
! rowspan="2" style="background: #D4E2EF;" | D108
 +
! rowspan="2" style="background: #D4E2EF;" | S03154
|align=center|2
|align=center|2
! rowspan="2"| XbaI
! rowspan="2"| XbaI
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|align=center| 3
|align=center| 3
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D109
 +
! rowspan="1" style="background: #D4E2EF;" | S03154
|align=center|4
|align=center|4
| align=center|EcoRI
| align=center|EcoRI
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|align=center| 4
|align=center| 4
|-
|-
-
! rowspan="4" style="background: #ccccff;" | D106
+
! rowspan="1" style="background: #D4E2EF;" | D110
-
! rowspan="4" style="background: #ccccff;" | S03879
+
! rowspan="1" style="background: #D4E2EF;" | S03879
|align=center|1
|align=center|1
| align=center|SpeI
| align=center|SpeI
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|align=center| 6
|align=center| 6
|-
|-
 +
! rowspan="2" style="background: #D4E2EF;" | D111
 +
! rowspan="2" style="background: #D4E2EF;" | S03879
|align=center|2
|align=center|2
! rowspan="2"| XbaI
! rowspan="2"| XbaI
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|align=center| 8
|align=center| 8
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D112
 +
! rowspan="1" style="background: #D4E2EF;" | S03879
|align=center|4
|align=center|4
| align=center|EcoRI
| align=center|EcoRI
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|align=center| 9
|align=center| 9
|-
|-
-
! rowspan="2" style="background: #ccccff;" |D107
+
! rowspan="1" style="background: #D4E2EF;" |D113
-
! rowspan="2" style="background: #ccccff;" |C0079
+
! rowspan="1" style="background: #D4E2EF;" |C0079
|align=center| 1
|align=center| 1
|align=center|EcoRI
|align=center|EcoRI
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|align=center| 10
|align=center| 10
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D114
 +
! rowspan="1" style="background: #D4E2EF;" | C0079
|align=center| 2
|align=center| 2
|align=center|XbaI
|align=center|XbaI
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|align=center| 11
|align=center| 11
|-
|-
-
! rowspan="2" style="background: #ccccff;"|D108
+
! rowspan="1" style="background: #D4E2EF;"|D115
-
! rowspan="2" style="background: #ccccff;"|C0179
+
! rowspan="1" style="background: #D4E2EF;"|C0179
|align=center| 1
|align=center| 1
|align=center| EcoRI
|align=center| EcoRI
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|align=center| 12
|align=center| 12
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D116
 +
! rowspan="1" style="background: #D4E2EF;" | C0179
|align=center| 2
|align=center| 2
|align=center| XbaI
|align=center| XbaI
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|align=center| 2
|align=center| 2
|-
|-
-
! rowspan="2" style="background: #ccccff;"|D109
+
! rowspan="1" style="background: #D4E2EF;"|D117
-
! rowspan="2" style="background: #ccccff;"|E0030
+
! rowspan="1" style="background: #D4E2EF;"|E0030
|align=center| 1
|align=center| 1
|align=center|EcoRI
|align=center|EcoRI
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|align=center| 3
|align=center| 3
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D118
 +
! rowspan="1" style="background: #D4E2EF;" | E0030
|align=center| 2
|align=center| 2
|align=center| XbaI
|align=center| XbaI
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|align=center| 4
|align=center| 4
|-
|-
-
! rowspan="2" style="background: #ccccff;"|D110
+
! rowspan="1" style="background: #D4E2EF;"|D119
-
! rowspan="2" style="background: #ccccff;"|E0040
+
! rowspan="1" style="background: #D4E2EF;"|E0040
|align=center| 1
|align=center| 1
|align=center| EcoRI
|align=center| EcoRI
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|align=center| 5
|align=center| 5
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D120
 +
! rowspan="1" style="background: #D4E2EF;" | E0040
|align=center| 2
|align=center| 2
|align=center| XbaI
|align=center| XbaI
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|align=center| 6
|align=center| 6
|-
|-
-
! rowspan="2" style="background: #ccccff;"|D111
+
! rowspan="1" style="background: #D4E2EF;"|D121
-
! rowspan="2" style="background: #ccccff;"|E1010
+
! rowspan="1" style="background: #D4E2EF;"|E1010
|align=center| 1
|align=center| 1
|align=center| EcoRI
|align=center| EcoRI
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|align=center| 7
|align=center| 7
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D122
 +
! rowspan="1" style="background: #D4E2EF;" | E1010
|align=center| 2
|align=center| 2
|align=center| XbaI
|align=center| XbaI
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|align=center| 8
|align=center| 8
|-
|-
-
! rowspan="2" style="background: #ccccff;"|D116
+
! rowspan="2" style="background: #D4E2EF;"|D123
-
! rowspan="2" style="background: #ccccff;"|J23100
+
! rowspan="2" style="background: #D4E2EF;"|J23100
|align=center|1
|align=center|1
! rowspan="2"| SpeI
! rowspan="2"| SpeI
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! rowspan="1"| 10
! rowspan="1"| 10
|-
|-
-
! rowspan="2" style="background: #ccccff;"|D117
+
! rowspan="2" style="background: #D4E2EF;"|D124
-
! rowspan="2" style="background: #ccccff;"|J23107
+
! rowspan="2" style="background: #D4E2EF;"|J23107
|align=center|1
|align=center|1
! rowspan="2"| SpeI
! rowspan="2"| SpeI
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! rowspan="1"| 12
! rowspan="1"| 12
|-
|-
-
! rowspan="2" style="background: #ccccff;"|D118
+
! rowspan="2" style="background: #D4E2EF;"|D125
-
! rowspan="2" style="background: #ccccff;"|B0015
+
! rowspan="2" style="background: #D4E2EF;"|B0015
|align=center|1
|align=center|1
! rowspan="2"|EcoRI
! rowspan="2"|EcoRI
! rowspan="2"|XbaI
! rowspan="2"|XbaI
-
! rowspan="2"| FV
+
! rowspan="2"| BV
! rowspan="2"| 3303 pb
! rowspan="2"| 3303 pb
! rowspan="2"| 15 pb
! rowspan="2"| 15 pb
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! rowspan="1"| 3
! rowspan="1"| 3
|-
|-
-
! rowspan="2" style="background: #ccccff;"|D119
+
! rowspan="2" style="background: #D4E2EF;"|D126
-
! rowspan="2" style="background: #ccccff;"|I0500
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! rowspan="2" style="background: #D4E2EF;"|I0500
|align=center|1
|align=center|1
! rowspan="2"|SpeI
! rowspan="2"|SpeI
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! rowspan="1"| 5
! rowspan="1"| 5
|-
|-
-
! rowspan="5" style="background: #ccccff;" | D120
+
! rowspan="2" style="background: #D4E2EF;" | D127
-
! rowspan="5" style="background: #ccccff;" | B0030
+
! rowspan="2" style="background: #D4E2EF;" | B0030
|align=center|1
|align=center|1
! rowspan="2"| XbaI
! rowspan="2"| XbaI
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! rowspan="1"| 5
! rowspan="1"| 5
|-
|-
 +
! rowspan="1" style="background: #D4E2EF;" | D128
 +
! rowspan="1" style="background: #D4E2EF;" | B0030
|align=center|3
|align=center|3
! rowspan="1"|  EcoRI
! rowspan="1"|  EcoRI
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! rowspan="1"| 6
! rowspan="1"| 6
|-
|-
 +
! rowspan="2" style="background: #D4E2EF;" | D129
 +
! rowspan="2" style="background: #D4E2EF;" | B0030
|align=center|4
|align=center|4
! rowspan="2"| SpeI
! rowspan="2"| SpeI
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! rowspan="1"| 8
! rowspan="1"| 8
|-
|-
-
! rowspan="3" style="background: #ccccff;"|D121
+
! rowspan="3" style="background: #D4E2EF;"|D130
-
! rowspan="3" style="background: #ccccff;"|E0422
+
! rowspan="3" style="background: #D4E2EF;"|E0422
|align=center|1
|align=center|1
! rowspan="3"| XbaI
! rowspan="3"| XbaI
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! rowspan="1"| 11
! rowspan="1"| 11
|-
|-
-
! rowspan="3" style="background: #ccccff;"|D122
+
! rowspan="3" style="background: #D4E2EF;"|D131
-
! rowspan="3" style="background: #ccccff;"|E0
+
! rowspan="3" style="background: #D4E2EF;"|E0840
|align=center|1
|align=center|1
! rowspan="3"| XbaI
! rowspan="3"| XbaI
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==> conclusion : all the digestion have succeed.....GREAT !
==> conclusion : all the digestion have succeed.....GREAT !
-
 
+
== DNA extraction ==
-
 
+
-
== Extraction of the DNA  ==
+

Latest revision as of 16:55, 31 July 2008

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Contents

MiniPreps

  • Use of Promega's protocol on all the clones cultivated on the 25th.
  • Preparation of 50µl of minipreps in double.


Name Biobrick Description
MP100 B0034 Strongest RBS (Efficiency = 1)
MP101 J23101 Strongest Constitutive promoter
MP102 J23109 Weak Constitutive promoter
MP103 R0079 promoter pLas (lasr & PAI regulated)
MP104 R0040 tetR repressible promoter
MP105 S03154 B0034(rbs) ->LasI
MP106 S03879 B0034(rbs) -> TetR
MP107 C0079 lasR activator with LVA Tag
MP108 C0179 lasR activator
MP109 E0030 YFP
MP110 E0040 GFP
MP111 E1010 mRFP
MP116 J23100 Strong constitutive promoter in J61002
MP117 J23107 Medium constitutive promoter in J61002
MP118 B0015 Double terminator
MP119 I0500 AraC pBAD
MP120 B0030 Strong RBS (Efficiency = 0,6)
MP121 E0422 ECFP (RBS+LVA+Term)
MP122 E0840 gfp tri-part; strong rbs


Digestion

Digestion Mix

10µl of Miniprep (26 aug.)
12.5µl of water
2.5µl of Buffer N°2
0.25µl of BSA 100x
1µl of enzyme 1
1µl of enzyme 2

  • Incubation 1h at 37°C with the first enzyme
  • Add the second enzyme
  • Incubation 1h at 37°C with the second enzyme
  • Store on ice
  • Revelation of the digestion by electrophoresis on agarose gel


Results of digestions : Electrophoresis

conditions :

  • 10µl of ladder 1 kb (except for gel n°6 : 100 pb)
  • 30µl of digestion added with 5µl of loading Dye 6x
  • migration ~30min at 100W
  • Gel 1, 2, 3, 4, 5 = 0.8%
  • Gel 6 = 1,5%


gel1 gel1 gel2 gel2 gel3 gel3 gel4 gel4


gel5 gel5 gel6 gel6


Name BioBrick Tube N° Enz 1 Enz 2 Obs Exp Size Matrix Exp Size BB Mea Size Matrix Mea Size BB Gel Band
D100 B0034 1 XbaI PstI BI 2057 pb 34 pb 1800 pb 40 pb 6 2
2 6 3
D101 B0034 3 EcoRI XbaI FV 2076 pb 15 pb 2000 pb - 1 2
D102 B0034 4 SpeI PstI BV 2077 pb 14 pb 2000 pb - 1 3
5 1 4
D103 J23101 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 700 pb 1 5
2 1 6
D104 J23109 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 700 pb 1 7
2 1 8
D105 R0079 1 SpeI PstI BV 2222 pb 14 pb 2000 pb - 1 9
2 1 10
D106 R0040 1 SpeI PstI BV 2119 pb 14 pb 2000 pb - 1 11
2 1 12
D107 S03154 1 SpeI PstI BV 2750 pb 14 pb 3000 pb - 2 5
D108 S03154 2 XbaI PstI BI 2057 pb 707 pb 2000 pb 700 pb 2 2
3 2 3
D109 S03154 4 EcoRI SpeI FI 2056 pb 708 pb 2000 pb 700 pb 2 4
D110 S03879 1 SpeI PstI BV 2768 pb 14 pb 2700 pb - 2 6
D111 S03879 2 XbaI PstI BI 2057 pb 725 pb 2000 pb 700 pb 2 7
3 2 8
D112 S03879 4 EcoRI SpeI FI 2756 pb 726 pb 3000 pb 700 pb 2 9
D113 C0079 1 EcoRI SpeI FI 4402 pb 779 pb 5000 pb 800 pb 2 10
D114 C0079 2 XbaI PstI BI 4003 pb 778 pb 5000 pb 800 pb 2 11
D115 C0179 1 EcoRI SpeI FI 4402 pb 746 pb 2000 pb 800 pb 2 12
D116 C0179 2 XbaI PstI BI 4403 pb 745 pb 2000 pb 800 pb 3 2
D117 E0030 1 EcoRI SpeI FI 3166 pb 746 pb 3000 pb 700 pb 3 3
D118 E0030 2 XbaI PstI BI 3167 pb 745 pb 3000 pb 700 pb 3 4
D119 E0040 1 EcoRI SpeI FI 2056 pb 743 pb 2000 pb 700 pb 3 5
D120 E0040 2 XbaI PstI BI 2057 pb 742 pb 2000 pb 700 pb 3 6
D121 E1010 1 EcoRI SpeI FI 4402 pb 704 pb 5000 pb 700 pb 3 7
D122 E1010 2 XbaI PstI BI 4403 pb 703 pb 5000 pb 700 pb 3 8
D123 J23100 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 900 pb 3 9
2 3 10
D124 J23107 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 900 pb 3 11
2 3 12
D125 B0015 1 EcoRI XbaI BV 3303 pb 15 pb 3000 pb - 4 2
2 4 3
D126 I0500 1 SpeI PstI FV 5621 pb 14 pb 6000 pb - 4 4
2 4 5
D127 B0030 1 XbaI PstI BI 2057 pb 37 pb 2000 pb 50 pb 6 4
2 6 5
D128 B0030 3 EcoRI XbaI FV 2079 pb 15 pb 2200 pb - 4 6
D129 B0030 4 SpeI PstI BV 2080 pb 14 pb 2000 pb - 4 7
5 4 8
D130 E0422 1 XbaI PstI BI 2057 pb 939 pb 2000 pb 1100 pb 4 9
2 4 10
3 4 11
D131 E0840 1 XbaI PstI BI 2057 pb 900 pb 2000 pb 1000 pb 4 12
2 2000 pb 900 pb 5 2
3 5 3


==> conclusion : all the digestion have succeed.....GREAT !

DNA extraction

  • Cutting of the parts of interest, for all the digestion that have migrated on the gels
  • Use of Promega's protocol for the extraction.
  • Test of the succeed of the extraction by electrophoresis on 2µl of the parts extracted.


==> conclusion : we succeed to detect DNA in our samples this time.

Retrieved from "http://2008.igem.org/Team:Paris/July_26"