Team:Paris/July 24

From 2008.igem.org

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== MiniPreps ==
== MiniPreps ==
-
*The protocol of MiniPrep of Promega has been done on all the clones cultivated on the 23th.
+
*The Promega MiniPreps protocol has been used on all the clones cultivated on the 23th.
{| border="1"  
{| border="1"  
Line 11: Line 11:
|align="center"|'''Description'''
|align="center"|'''Description'''
|-
|-
-
|align="center"|MP116 (=MP1)
+
|align="center"|MP116
-
|align="center"|J23100
+
|align="center"|[http://partsregistry.org/Part:BBa_J23100 J23100]
-
|align="center"|
+
|align="center"|Strong constitutive promoter in J61002
|-
|-
-
|align="center"|MP117 (=MP2)
+
|align="center"|MP117
-
|align="center"|J23107
+
|align="center"|[http://partsregistry.org/Part:BBa_J23107 J23107]
-
|align="center"|
+
|align="center"|Medium constitutive promoter in J61002
|-
|-
-
|align="center"|MP118 (=MP3)
+
|align="center"|MP118
-
|align="center"|B0015
+
|align="center"|[http://partsregistry.org/Part:BBa_B0015 B0015]
-
|align="center"|
+
|align="center"|Double terminator
|-
|-
-
|align="center"|MP119 (=MP4)
+
|align="center"|MP119
-
|align="center"|I0500
+
|align="center"|[http://partsregistry.org/Part:BBa_I0500 I0500]
-
|align="center"|
+
|align="center"|AraC pBAD
|-
|-
-
|align="center"|MP120 (=MP5)
+
|align="center"|MP120
-
|align="center"|B0030
+
|align="center"|[http://partsregistry.org/Part:BBa_B0030 B0030]
-
|align="center"|
+
|align="center"|Strong RBS (Efficiency = 0,6)
|-
|-
-
|align="center"|MP121 (=MP6)
+
|align="center"|MP121
-
|align="center"|E0422
+
|align="center"|[http://partsregistry.org/Part:BBa_E0422 E0422]
-
|align="center"|
+
|align="center"|ECFP (RBS+LVA+Term)
|-
|-
-
|align="center"|MP122 (=MP8)
+
|align="center"| MP122
-
|align="center"|E0840
+
|align="center"|[http://partsregistry.org/Part:BBa_E0840 E0840]
-
|align="center"|
+
|align="center"|gfp tri-part; strong rbs
 +
|
|}
|}
 +
== Digestion ==
 +
===Digestion Mix===
-
== Digestion ==
+
10µl of Miniprep (26 aug.) <br>
 +
12.5µl of water<br>
 +
2.5µl of Buffer N°2<br>
 +
0.25µl of BSA 100x<br>
 +
1µl of enzyme 1<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
-
===Digestion Mix===
 
-
10µl of Miniprep (clone 1 or 2)
+
=== Results of digestions : Electrophoresis ===
-
12.5µl of water
+
 
-
2.5µl of Buffer N°2
+
conditions :
-
0.25µl of BSA 100x
+
* 10µl of ladder 1 kb (except for gel n°7 : 100 pb)
-
1µl of enzyme 1
+
* 30µl of digestion added with 5µl of loading Dye 6x
-
1µl of enzyme 2
+
* migration ~30min at 100W
 +
* Gel 1, 2, 3, 4, 5, 6, 8 = '''0.8%'''   
 +
* Gel 7 = '''1,2%'''
 +
 
 +
 
 +
 
 +
gel1 [[Image:Gel1.jpg| gel1|200px]]
 +
gel2 [[Image:Gel2.jpg| gel2|200px]]
 +
gel3 [[Image:Gel3.jpg| gel3|200px]]
 +
gel4 [[Image:Gel4.jpg| gel4|200px]]<br>
 +
 
 +
 
 +
gel5 [[Image:Gel_5.jpg‎| gel5|200px]]
 +
gel6 [[Image:Gel_6.jpg‎| gel6|200px]]
 +
gel7 [[Image:Gel_7.jpg‎| gel7|100px]]
 +
gel8 [[Image:Gel_8.jpg‎| gel8|100px]]
-
===list of digestion===
 
{| border="1"  
{| border="1"  
-
|align="center"|'''Name'''
+
|align=center|'''Name'''
-
|align="center"|'''Miniprep'''
+
|align=center|'''BioBrick'''
-
|align="center"|'''Biobrick'''
+
|align=center|'''Tube N°'''
-
|align="center"|'''Enzyme 1'''
+
|align=center|'''Enz 1'''
-
|align="center"|'''Enzyme 2'''
+
|align=center|'''Enz 2'''
-
|align="center"|'''Nber tube'''
+
|align=center|'''Obs'''
 +
|align=center|'''Exp Size Matrix'''
 +
|align=center|'''Exp Size BB'''
 +
|align=center|'''Mea Size Matrix'''
 +
|align=center|'''Mea Size BB'''
 +
|align=center|'''Gel'''
 +
|align=center|'''Band'''
|-
|-
-
|align="center"|D100
+
! rowspan="5" style="background: #ccccff;" | D100
-
|align="center"|MP100
+
! rowspan="5" style="background: #ccccff;" | B0034
-
|align="center"|B0034
+
|align=center|1
-
|align="center"|XbaI
+
! rowspan="2"| XbaI
-
|align="center"|PstI
+
! rowspan="2"| PstI
-
|align="center"|2
+
! rowspan="2"| BI
 +
! rowspan="2"| 2057 pb
 +
! rowspan="2"| 34 pb
 +
! rowspan="2"| not digested
 +
! rowspan="2"| not digested
 +
! rowspan="2"| 8
 +
! rowspan="2"| 5-6
|-
|-
-
|align="center"|
+
|align=center|2
-
|align="center"|
+
-
|align="center"|
+
-
|align="center"|EcoRI
+
-
|align="center"|XbaI
+
-
|align="center"|1
+
|-
|-
-
|align="center"|
+
|align=center|3
-
|align="center"|
+
|align=center| EcoRI
-
|align="center"|
+
|align=center| XbaI
-
|align="center"|SpeI
+
|align=center| FV
-
|align="center"|PstI
+
|align=center| 2076 pb
-
|align="center"|2
+
|align=center| 15 pb
 +
|align=center| 2100 pb
 +
|align=center| -
 +
|align=center| 1
 +
|align=center| 2
|-
|-
-
|align="center"|D101
+
|align=center|4
-
|align="center"|MP101
+
! rowspan="2"| SpeI
-
|align="center"|J23101
+
! rowspan="2"| PstI
-
|align="center"|SpeI
+
! rowspan="2"| BV
-
|align="center"|PstI
+
! rowspan="2"| 2077 pb
-
|align="center"|2
+
! rowspan="2"| 14 pb
 +
! rowspan="2"| 2000 pb
 +
! rowspan="2"| -
 +
|align=center| 1
 +
|align=center| 3-4
|-
|-
-
|align="center"|D102
+
|align=center|5
-
|align="center"|MP102
+
|align=center| 1
-
|align="center"|J23109
+
|align=center| 5-6
-
|align="center"|SpeI
+
-
|align="center"|PstI
+
-
|align="center"|2
+
|-
|-
-
|align="center"|D103
+
! rowspan="2" style="background: #ccccff;" | D101
-
|align="center"|MP103
+
! rowspan="2" style="background: #ccccff;" | J23101
-
|align="center"|R0079
+
|align=center|1
-
|align="center"|SpeI
+
! rowspan="2"| SpeI
-
|align="center"|PstI
+
! rowspan="2"| PstI
-
|align="center"|2
+
! rowspan="2"| BV
 +
! rowspan="2"| 2100 pb
 +
! rowspan="2"| 883 pb
 +
! rowspan="2"| 2000 pb
 +
! rowspan="2"| 900 pb
 +
|align=center| 1
 +
|align=center| 7-8
|-
|-
-
|align="center"|D104
+
|align=center|2
-
|align="center"|MP104
+
|align=center| 1
-
|align="center"|R0040
+
|align=center| 9-10
-
|align="center"|SpeI
+
-
|align="center"|PstI
+
-
|align="center"|2
+
|-
|-
-
|align="center"|D105
+
! rowspan="1" style="background: #ccccff;" | D102
-
|align="center"|MP105
+
! rowspan="1" style="background: #ccccff;" | J23109
-
|align="center"|S03154
+
|align=center|1
-
|align="center"|SpeI
+
! rowspan="1"| SpeI
-
|align="center"|PstI
+
! rowspan="1"| PstI
-
|align="center"|1
+
! rowspan="1"| BV
 +
! rowspan="1"| 2100 pb
 +
! rowspan="1"| 883 pb
 +
|align=center| 2000 pb
 +
|align=center| 900 pb
 +
|align=center| 1 & 2
 +
|align=center| 11-12 & 2-3
|-
|-
-
|align="center"|
+
! rowspan="2" style="background: #ccccff;" | D103
-
|align="center"|
+
! rowspan="2" style="background: #ccccff;" | R0079
-
|align="center"|
+
|align=center|1
-
|align="center"|XbaI
+
! rowspan="2"| SpeI
-
|align="center"|PstI
+
! rowspan="2"| PstI
-
|align="center"|2
+
! rowspan="2"| BV
 +
! rowspan="2"| 2222 pb
 +
! rowspan="2"| 14 pb
 +
! rowspan="2"| 2500 pb
 +
! rowspan="2"| -
 +
|align=center| 2
 +
|align=center| 4-5
|-
|-
-
|align="center"|
+
|align=center|2
-
|align="center"|
+
|align=center| 2
-
|align="center"|
+
|align=center| 6-7
-
|align="center"|EcoRI
+
-
|align="center"|SpeI
+
-
|align="center"|1
+
|-
|-
-
|align="center"|D106
+
! rowspan="2" style="background: #ccccff;" | D104
-
|align="center"|MP106
+
! rowspan="2" style="background: #ccccff;" | R0040
-
|align="center"|J23101
+
|align=center|1
-
|align="center"|SpeI
+
! rowspan="2"| SpeI
-
|align="center"|PstI
+
! rowspan="2"| PstI
-
|align="center"|1
+
! rowspan="2"| BV
 +
! rowspan="2"| 2119 pb
 +
! rowspan="2"| 14 pb
 +
! rowspan="2"| 2500 pb
 +
! rowspan="2"| -
 +
|align=center| 2
 +
|align=center| 8-9
|-
|-
-
|align="center"|
+
|align=center|2
-
|align="center"|
+
|align=center| 2
-
|align="center"|
+
|align=center| 10-11
-
|align="center"|XbaI
+
-
|align="center"|PstI
+
-
|align="center"|2
+
|-
|-
-
|align="center"|
+
! rowspan="4" style="background: #ccccff;" | D105
-
|align="center"|
+
! rowspan="4" style="background: #ccccff;" | S03154
-
|align="center"|
+
|align=center|1
-
|align="center"|EcoRI
+
! rowspan="1"| SpeI
-
|align="center"|SpeI
+
! rowspan="1"| PstI
-
|align="center"|1
+
! rowspan="1"| BV
 +
! rowspan="1"| 2750 pb
 +
! rowspan="1"| 14 pb
 +
|align=center| 3000 pb
 +
|align=center| -
 +
|align=center| 2 & 3
 +
|align=center| 12 & 2
|-
|-
-
|align="center"|D107
+
|align=center|2
-
|align="center"|MP107
+
! rowspan="2"| XbaI
-
|align="center"|C0079
+
! rowspan="2"| PstI
-
|align="center"|EcoRI
+
! rowspan="2"| BI
-
|align="center"|SpeI
+
! rowspan="2"| 2057 pb
-
|align="center"|1
+
! rowspan="2"| 707 pb
 +
! rowspan="2"| 2000 pb
 +
! rowspan="2"| 700 pb
 +
|align=center| 3
 +
|align=center| 3-4
|-
|-
-
|align="center"|D108
+
|align=center|3
-
|align="center"|MP108
+
|align=center| 3
-
|align="center"|C0179
+
|align=center| 5-6
-
|align="center"|EcoRI
+
-
|align="center"|SpeI
+
-
|align="center"|1
+
|-
|-
-
|align="center"|D109
+
|align=center|4
-
|align="center"|MP109
+
| align=center|EcoRI
-
|align="center"|E0030
+
| align=center|SpeI
-
|align="center"|EcoRI
+
| align=center|FI
-
|align="center"|SpeI
+
| align=center| 2056 pb
-
|align="center"|1
+
| align=center| 708 pb
 +
|align=center| 2000 pb
 +
|align=center| 700 pb
 +
|align=center| 3
 +
|align=center| 7-8
|-
|-
-
|align="center"|D110
+
! rowspan="4" style="background: #ccccff;" | D106
-
|align="center"|MP110
+
! rowspan="4" style="background: #ccccff;" | S03879
-
|align="center"|E0040
+
|align=center|1
-
|align="center"|EcoRI
+
| align=center|SpeI
-
|align="center"|SpeI
+
| align=center|PstI
-
|align="center"|1
+
| align=center|BV
 +
| align=center| 2768 pb
 +
| align=center|14 pb
 +
|align=center| 3000 pb
 +
|align=center| -
 +
|align=center| 3
 +
|align=center| 9-10
|-
|-
-
|align="center"|D111
+
|align=center|2
-
|align="center"|MP111
+
! rowspan="2"| XbaI
-
|align="center"|E1010
+
! rowspan="2"| PstI
-
|align="center"|EcoRI
+
! rowspan="2"| BI
-
|align="center"|SpeI
+
! rowspan="2"| 2057 pb
-
|align="center"|1
+
! rowspan="2"| 725 pb
 +
! rowspan="2"| 2000 pb
 +
! rowspan="2"| 700 pb
 +
|align=center| 3
 +
|align=center| 11-12
|-
|-
-
|align="center"|D116
+
|align=center|3
-
|align="center"|MP116
+
|align=center| 4
-
|align="center"|J23100
+
|align=center| 2-3
-
|align="center"|SpeI
+
-
|align="center"|PstI
+
-
|align="center"|2
+
|-
|-
-
|align="center"|D117
+
|align=center|4
-
|align="center"|MP117
+
| align=center|EcoRI
-
|align="center"|J23107
+
| align=center|SpeI
-
|align="center"|SpeI
+
| align=center|FI
-
|align="center"|PstI
+
| align=center| 2756 pb
-
|align="center"|2
+
| align=center| 726 pb
 +
|align=center|  2500 pb
 +
|align=center| 700 pb
 +
|align=center| 4
 +
|align=center| 4-5
|-
|-
-
|align="center"|D118
+
! rowspan="2" style="background: #ccccff;" |D107
-
|align="center"|MP118
+
! rowspan="2" style="background: #ccccff;" |C0079
-
|align="center"|B0015
+
|align=center| 1
-
|align="center"|EcoRI
+
|align=center|EcoRI
-
|align="center"|XbaI
+
|align=center|SpeI
-
|align="center"|2
+
|align=center|FI
 +
|align=center| 4402 pb
 +
|align=center| 779 pb
 +
|align=center| 6000 pb
 +
|align=center| 1000 pb
 +
|align=center| 4
 +
|align=center| 6-7
|-
|-
-
|align="center"|D119
+
|align=center| 2
-
|align="center"|MP119
+
|align=center|XbaI
-
|align="center"|I0500
+
|align=center|PstI
-
|align="center"|SpeI
+
|align=center|BI
-
|align="center"|PstI
+
|align=center| 4003 pb
-
|align="center"|2
+
|align=center| 778 pb
 +
|align=center| 5000 pb
 +
|align=center| 100 pb
 +
|align=center| 6
 +
|align=center| 9
|-
|-
-
|align="center"|D120
+
! rowspan="2" style="background: #ccccff;"|D108
-
|align="center"|MP120
+
! rowspan="2" style="background: #ccccff;"|C0179
-
|align="center"|B0030
+
|align=center| 1
-
|align="center"|SpeI
+
|align=center| EcoRI
-
|align="center"|PstI
+
|align=center| SpeI
-
|align="center"|2
+
|align=center| FI
 +
|align=center| 4402 pb
 +
|align=center| 746 pb
 +
|align=center| 2500 pb
 +
|align=center| 1000 pb
 +
|align=center| 4
 +
|align=center| 8-9
|-
|-
-
|align="center"|
+
|align=center| 2
-
|align="center"|
+
|align=center| XbaI
-
|align="center"|
+
|align=center| PstI
-
|align="center"|EcoRI
+
|align=center| BI
-
|align="center"|XbaI
+
|align=center| 4403 pb
-
|align="center"|1
+
|align=center| 745 pb
 +
|align=center| 2500 pb
 +
|align=center| 1000 pb
 +
|align=center| 6
 +
|align=center| 10
|-
|-
-
|align="center"|
+
! rowspan="2" style="background: #ccccff;"|D109
-
|align="center"|
+
! rowspan="2" style="background: #ccccff;"|E0030
-
|align="center"|
+
|align=center| 1
-
|align="center"|XbaI
+
|align=center|EcoRI
-
|align="center"|PstI
+
|align=center|SpeI
-
|align="center"|2
+
|align=center|FI
 +
|align=center| 3166 pb
 +
|align=center| 746 pb
 +
|align=center| 4000 pb
 +
|align=center| 1000 pb
 +
|align=center| 4
 +
|align=center| 10-11
|-
|-
-
|align="center"|D121
+
|align=center| 2
-
|align="center"|MP121
+
|align=center| XbaI
-
|align="center"|E0422
+
|align=center| PstI
-
|align="center"|XbaI
+
|align=center| BI
-
|align="center"|PstI
+
|align=center| 3167 pb
-
|align="center"|3
+
|align=center| 745 pb
 +
|align=center| 3000 pb
 +
|align=center| 1000 pb
 +
|align=center| 6
 +
|align=center| 11
|-
|-
-
|align="center"|D122
+
! rowspan="2" style="background: #ccccff;"|D110
-
|align="center"|MP122
+
! rowspan="2" style="background: #ccccff;"|E0040
-
|align="center"|E0840
+
|align=center| 1
-
|align="center"|XbaI
+
|align=center| EcoRI
-
|align="center"|PstI
+
|align=center| SpeI
-
|align="center"|3
+
|align=center| FI
 +
|align=center| 2056 pb
 +
|align=center| 743 pb
 +
|align=center| 2000 pb
 +
|align=center| 800 pb
 +
|align=center| 4 & 5
 +
|align=center| 12 & 2
 +
|-
 +
|align=center| 2
 +
|align=center| XbaI
 +
|align=center| PstI
 +
|align=center| BI
 +
|align=center| 2057 pb
 +
|align=center| 742 pb
 +
|align=center| 2500 pb
 +
|align=center| 1000 pb
 +
|align=center| 6
 +
|align=center| 12
 +
|-
 +
! rowspan="2" style="background: #ccccff;"|D111
 +
! rowspan="2" style="background: #ccccff;"|E1010
 +
|align=center| 1
 +
|align=center| EcoRI
 +
|align=center| SpeI
 +
|align=center| FI
 +
|align=center| 4402 pb
 +
|align=center| 704 pb
 +
|align=center| 4000 pb
 +
|align=center| 600 pb
 +
|align=center| 5
 +
|align=center| 3-4
 +
|-
 +
|align=center| 2
 +
|align=center| XbaI
 +
|align=center| PstI
 +
|align=center| BI
 +
|align=center| 4403 pb
 +
|align=center| 703 pb
 +
|align=center| 4500 pb
 +
|align=center| 700 pb
 +
|align=center| 8
 +
|align=center| 2-3
 +
|-
 +
! rowspan="1" style="background: #ccccff;"|D116
 +
! rowspan="1" style="background: #ccccff;"|J23100
 +
|align=center|1
 +
! rowspan="1"| SpeI
 +
! rowspan="1"| PstI
 +
! rowspan="1"| BV
 +
! rowspan="1"| 2100 pb
 +
! rowspan="1"| 883 pb
 +
! colspan="4"| Not realised
 +
|align=center|
 +
|-
 +
! rowspan="1" style="background: #ccccff;"|D117
 +
! rowspan="1" style="background: #ccccff;"|J23107
 +
|align=center|1
 +
! rowspan="1"| SpeI
 +
! rowspan="1"| PstI
 +
! rowspan="1"| BV
 +
! rowspan="1"| 2100 pb
 +
! rowspan="1"| 883 pb
 +
|align=center| 2000 pb
 +
|align=center| 800 pb
 +
|align=center| 5
 +
|align=center| 5-6
 +
|-
 +
! rowspan="1" style="background: #ccccff;"|D118
 +
! rowspan="1" style="background: #ccccff;"|B0015
 +
|align=center|1
 +
|align=center|EcoRI
 +
|align=center|XbaI
 +
! rowspan="1"| FV
 +
! rowspan="1"| 3303 pb
 +
! rowspan="1"| 15 pb
 +
|align=center| 3000 pb
 +
|align=center| -
 +
|align=center| 5
 +
|align=center| 7-8
 +
|-
 +
! rowspan="1" style="background: #ccccff;"|D119
 +
! rowspan="1" style="background: #ccccff;"|I0500
 +
|align=center|1
 +
|align=center|SpeI
 +
|align=center|PstI
 +
! rowspan="1"| FV
 +
! rowspan="1"| 5621 pb
 +
! rowspan="1"| 14 pb
 +
|align=center| 6000 - 3000 pb
 +
|align=center| -
 +
|align=center| 5
 +
|align=center| 9-10
 +
|-
 +
! rowspan="5" style="background: #ccccff;" | D120
 +
! rowspan="5" style="background: #ccccff;" | B0030
 +
|align=center|1
 +
! rowspan="2"| XbaI
 +
! rowspan="2"| PstI
 +
! rowspan="2"| BI
 +
! rowspan="2"| 2057 pb
 +
! rowspan="2"| 37 pb
 +
! rowspan="2"| not digested
 +
! rowspan="2"| not digested
 +
|align=center| 7
 +
|align=center| 2
 +
|-
 +
|align=center|2
 +
|align=center| 7
 +
|align=center| 5-6
 +
|-
 +
|align=center|3
 +
|align=center| EcoRI
 +
|align=center| XbaI
 +
|align=center| FV
 +
|align=center| 2079 pb
 +
|align=center| 15 pb
 +
|align=center| 1600 pb
 +
|align=center| -
 +
|align=center| 6
 +
|align=center| 2
 +
|-
 +
|align=center|4
 +
! rowspan="2"| SpeI
 +
! rowspan="2"| PstI
 +
! rowspan="2"| BV
 +
! rowspan="2"| 2080 pb
 +
! rowspan="2"| 14 pb
 +
! rowspan="2"| 3000 - 2000 pb
 +
! rowspan="2"| -
 +
|align=center| 5
 +
|align=center| 11
 +
|-
 +
|align=center|5
 +
|align=center| 5
 +
|align=center| 12
 +
|-
 +
! rowspan="1" style="background: #ccccff;"|D121
 +
! rowspan="1" style="background: #ccccff;"|E0422
 +
|align=center|1
 +
|align=center|XbaI
 +
|align=center|PstI
 +
|align=center| FV
 +
|align=center| 2057 pb
 +
|align=center| 939 pb
 +
|align=center| 3000 pb
 +
|align=center| Not digested
 +
|align=center| 6
 +
|align=center| 3-5
 +
|-
 +
! rowspan="1" style="background: #ccccff;"|D122
 +
! rowspan="1" style="background: #ccccff;"|E0840
 +
|align=center|1
 +
|align=center|XbaI
 +
|align=center|PstI
 +
|align=center| FV
 +
|align=center| 2057 pb
 +
|align=center| 900 pb
 +
|align=center| 2500 pb
 +
|align=center| Not digested
 +
|align=center| 6
 +
|align=center| 6-8
|}
|}
 +
 +
 +
==> Conclusion: Most of the digestion have succeed
 +
 +
== Extraction of the DNA  ==
 +
 +
 +
* Cutting of the parts of interest, for all the digestion that have migrated on the gels
 +
* Store of all the piece of gel O/N at -20°C.

Latest revision as of 14:39, 31 July 2008

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Contents

MiniPreps

  • The Promega MiniPreps protocol has been used on all the clones cultivated on the 23th.
Name Biobrick Description
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°7 : 100 pb)
  • 30µl of digestion added with 5µl of loading Dye 6x
  • migration ~30min at 100W
  • Gel 1, 2, 3, 4, 5, 6, 8 = 0.8%
  • Gel 7 = 1,2%


gel1 gel1 gel2 gel2 gel3 gel3 gel4 gel4


gel5 gel5 gel6 gel6 gel7 gel7 gel8 gel8


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 not digested not digested 8 5-6
2
3 EcoRI XbaI FV 2076 pb 15 pb 2100 pb - 1 2
4 SpeI PstI BV 2077 pb 14 pb 2000 pb - 1 3-4
5 1 5-6
D101 J23101 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 900 pb 1 7-8
2 1 9-10
D102 J23109 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 900 pb 1 & 2 11-12 & 2-3
D103 R0079 1 SpeI PstI BV 2222 pb 14 pb 2500 pb - 2 4-5
2 2 6-7
D104 R0040 1 SpeI PstI BV 2119 pb 14 pb 2500 pb - 2 8-9
2 2 10-11
D105 S03154 1 SpeI PstI BV 2750 pb 14 pb 3000 pb - 2 & 3 12 & 2
2 XbaI PstI BI 2057 pb 707 pb 2000 pb 700 pb 3 3-4
3 3 5-6
4 EcoRI SpeI FI 2056 pb 708 pb 2000 pb 700 pb 3 7-8
D106 S03879 1 SpeI PstI BV 2768 pb 14 pb 3000 pb - 3 9-10
2 XbaI PstI BI 2057 pb 725 pb 2000 pb 700 pb 3 11-12
3 4 2-3
4 EcoRI SpeI FI 2756 pb 726 pb 2500 pb 700 pb 4 4-5
D107 C0079 1 EcoRI SpeI FI 4402 pb 779 pb 6000 pb 1000 pb 4 6-7
2 XbaI PstI BI 4003 pb 778 pb 5000 pb 100 pb 6 9
D108 C0179 1 EcoRI SpeI FI 4402 pb 746 pb 2500 pb 1000 pb 4 8-9
2 XbaI PstI BI 4403 pb 745 pb 2500 pb 1000 pb 6 10
D109 E0030 1 EcoRI SpeI FI 3166 pb 746 pb 4000 pb 1000 pb 4 10-11
2 XbaI PstI BI 3167 pb 745 pb 3000 pb 1000 pb 6 11
D110 E0040 1 EcoRI SpeI FI 2056 pb 743 pb 2000 pb 800 pb 4 & 5 12 & 2
2 XbaI PstI BI 2057 pb 742 pb 2500 pb 1000 pb 6 12
D111 E1010 1 EcoRI SpeI FI 4402 pb 704 pb 4000 pb 600 pb 5 3-4
2 XbaI PstI BI 4403 pb 703 pb 4500 pb 700 pb 8 2-3
D116 J23100 1 SpeI PstI BV 2100 pb 883 pb Not realised
D117 J23107 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 800 pb 5 5-6
D118 B0015 1 EcoRI XbaI FV 3303 pb 15 pb 3000 pb - 5 7-8
D119 I0500 1 SpeI PstI FV 5621 pb 14 pb 6000 - 3000 pb - 5 9-10
D120 B0030 1 XbaI PstI BI 2057 pb 37 pb not digested not digested 7 2
2 7 5-6
3 EcoRI XbaI FV 2079 pb 15 pb 1600 pb - 6 2
4 SpeI PstI BV 2080 pb 14 pb 3000 - 2000 pb - 5 11
5 5 12
D121 E0422 1 XbaI PstI FV 2057 pb 939 pb 3000 pb Not digested 6 3-5
D122 E0840 1 XbaI PstI FV 2057 pb 900 pb 2500 pb Not digested 6 6-8


==> Conclusion: Most of the digestion have succeed

Extraction of the DNA

  • Cutting of the parts of interest, for all the digestion that have migrated on the gels
  • Store of all the piece of gel O/N at -20°C.
Retrieved from "http://2008.igem.org/Team:Paris/July_24"