Team:Paris/July 26

From 2008.igem.org

(Difference between revisions)
(→Extraction of the DNA)
 
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== MiniPreps ==
== MiniPreps ==
-
*The protocol of MiniPrep of Promega has been done on all the clones cultivated on the 25th.
+
 
 +
* Use of Promega's protocol on all the clones cultivated on the 25th.
 +
* Preparation of 50µl of minipreps in double.
 +
 
{| border="1"  
{| border="1"  
Line 96: Line 99:
===Digestion Mix===
===Digestion Mix===
-
10µl of Miniprep (clone 1 or 2)<br>
+
10µl of Miniprep (26 aug.) <br>
12.5µl of water<br>
12.5µl of water<br>
2.5µl of Buffer N°2<br>
2.5µl of Buffer N°2<br>
Line 102: Line 105:
1µl of enzyme 1<br>
1µl of enzyme 1<br>
1µl of enzyme 2<br>
1µl of enzyme 2<br>
 +
 +
* 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 ===
=== 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 115: 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
+
! 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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! rowspan="1"| 3
! rowspan="1"| 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.

Latest revision as of 16:55, 31 July 2008

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↓ Calendar ↑

Tomorrow →


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 [http://partsregistry.org/Part:BBa_B0034 B0034] Strongest RBS (Efficiency = 1)
MP101 [http://partsregistry.org/Part:BBa_J23101 J23101] Strongest Constitutive promoter
MP102 [http://partsregistry.org/Part:BBa_J23109 J23109] Weak Constitutive promoter
MP103 [http://partsregistry.org/Part:BBa_R0079 R0079] promoter pLas (lasr & PAI regulated)
MP104 [http://partsregistry.org/Part:BBa_R0040 R0040] tetR repressible promoter
MP105 [http://partsregistry.org/Part:BBa_S03154 S03154] B0034(rbs) ->LasI
MP106 [http://partsregistry.org/Part:BBa_S03879 S03879] B0034(rbs) -> TetR
MP107 [http://partsregistry.org/Part:BBa_C0079 C0079] lasR activator with LVA Tag
MP108 [http://partsregistry.org/Part:BBa_C0179 C0179] lasR activator
MP109 [http://partsregistry.org/Part:BBa_E0030 E0030] YFP
MP110 [http://partsregistry.org/Part:BBa_E0040 E0040] GFP
MP111 [http://partsregistry.org/Part:BBa_E1010 E1010] mRFP
MP116 [http://partsregistry.org/Part:BBa_J23100 J23100] Strong constitutive promoter in J61002
MP117 [http://partsregistry.org/Part:BBa_J23107 J23107] Medium constitutive promoter in J61002
MP118 [http://partsregistry.org/Part:BBa_B0015 B0015] Double terminator
MP119 [http://partsregistry.org/Part:BBa_I0500 I0500] AraC pBAD
MP120 [http://partsregistry.org/Part:BBa_B0030 B0030] Strong RBS (Efficiency = 0,6)
MP121 [http://partsregistry.org/Part:BBa_E0422 E0422] ECFP (RBS+LVA+Term)
MP122 [http://partsregistry.org/Part:BBa_E0840 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"