Team:Paris/July 24

From 2008.igem.org

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(→MiniPreps)
 
(23 intermediate revisions not shown)
Line 4: Line 4:
== 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 ==
-
 
===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 56: Line 53:
1µl of enzyme 2<br>
1µl of enzyme 2<br>
-
===list of digestion===
+
* 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
-
{| border="1"
 
-
|align="center"|'''Name'''
 
-
|align="center"|'''Miniprep'''
 
-
|align="center"|'''Biobrick'''
 
-
|align="center"|'''Enzyme 1'''
 
-
|align="center"|'''Enzyme 2'''
 
-
|align="center"|'''Nber tube'''
 
-
|-
 
-
|align="center"|D100
 
-
|align="center"|MP100
 
-
|align="center"|B0034
 
-
|align="center"|XbaI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|EcoRI
 
-
|align="center"|XbaI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D101
 
-
|align="center"|MP101
 
-
|align="center"|J23101
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D102
 
-
|align="center"|MP102
 
-
|align="center"|J23109
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D103
 
-
|align="center"|MP103
 
-
|align="center"|R0079
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D104
 
-
|align="center"|MP104
 
-
|align="center"|R0040
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D105
 
-
|align="center"|MP105
 
-
|align="center"|S03154
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|XbaI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|EcoRI
 
-
|align="center"|SpeI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|D106
 
-
|align="center"|MP106
 
-
|align="center"|J23101
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|XbaI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|EcoRI
 
-
|align="center"|SpeI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|D107
 
-
|align="center"|MP107
 
-
|align="center"|C0079
 
-
|align="center"|EcoRI
 
-
|align="center"|SpeI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|D108
 
-
|align="center"|MP108
 
-
|align="center"|C0179
 
-
|align="center"|EcoRI
 
-
|align="center"|SpeI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|D109
 
-
|align="center"|MP109
 
-
|align="center"|E0030
 
-
|align="center"|EcoRI
 
-
|align="center"|SpeI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|D110
 
-
|align="center"|MP110
 
-
|align="center"|E0040
 
-
|align="center"|EcoRI
 
-
|align="center"|SpeI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|D111
 
-
|align="center"|MP111
 
-
|align="center"|E1010
 
-
|align="center"|EcoRI
 
-
|align="center"|SpeI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|D116
 
-
|align="center"|MP116
 
-
|align="center"|J23100
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D117
 
-
|align="center"|MP117
 
-
|align="center"|J23107
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D118
 
-
|align="center"|MP118
 
-
|align="center"|B0015
 
-
|align="center"|EcoRI
 
-
|align="center"|XbaI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D119
 
-
|align="center"|MP119
 
-
|align="center"|I0500
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D120
 
-
|align="center"|MP120
 
-
|align="center"|B0030
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|EcoRI
 
-
|align="center"|XbaI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|XbaI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D121
 
-
|align="center"|MP121
 
-
|align="center"|E0422
 
-
|align="center"|XbaI
 
-
|align="center"|PstI
 
-
|align="center"|3
 
-
|-
 
-
|align="center"|D122
 
-
|align="center"|MP122
 
-
|align="center"|E0840
 
-
|align="center"|XbaI
 
-
|align="center"|PstI
 
-
|align="center"|3
 
-
|}
 
-
== Results of digestions : Electrophoresis ==
+
=== 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 [[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]]
 +
 
 +
 
{| border="1"  
{| border="1"  
Line 270: Line 96:
|align=center|'''Mea Size Matrix'''
|align=center|'''Mea Size Matrix'''
|align=center|'''Mea Size BB'''
|align=center|'''Mea Size BB'''
 +
|align=center|'''Gel'''
 +
|align=center|'''Band'''
|-
|-
! rowspan="5" style="background: #ccccff;" | D100
! rowspan="5" style="background: #ccccff;" | D100
Line 281: Line 109:
! rowspan="2"| not digested
! rowspan="2"| not digested
! rowspan="2"| not digested
! rowspan="2"| not digested
 +
! rowspan="2"| 8
 +
! rowspan="2"| 5-6
|-
|-
|align=center|2
|align=center|2
-
|align=center|
 
-
|align=center|
 
|-
|-
|align=center|3
|align=center|3
Line 294: Line 122:
|align=center| 2100 pb  
|align=center| 2100 pb  
|align=center| -
|align=center| -
 +
|align=center| 1
 +
|align=center| 2
|-
|-
|align=center|4
|align=center|4
Line 303: Line 133:
! rowspan="2"| 2000 pb
! rowspan="2"| 2000 pb
! rowspan="2"| -
! rowspan="2"| -
 +
|align=center| 1
 +
|align=center| 3-4
|-
|-
|align=center|5
|align=center|5
-
|align=center|
+
|align=center| 1
-
|align=center|
+
|align=center| 5-6
|-
|-
! rowspan="2" style="background: #ccccff;" | D101
! rowspan="2" style="background: #ccccff;" | D101
Line 318: Line 150:
! rowspan="2"| 2000 pb
! rowspan="2"| 2000 pb
! rowspan="2"| 900 pb
! rowspan="2"| 900 pb
 +
|align=center| 1
 +
|align=center| 7-8
|-
|-
|align=center|2
|align=center|2
-
|align=center|
+
|align=center| 1
-
|align=center|
+
|align=center| 9-10
|-
|-
! rowspan="1" style="background: #ccccff;" | D102
! rowspan="1" style="background: #ccccff;" | D102
Line 333: Line 167:
|align=center| 2000 pb
|align=center| 2000 pb
|align=center| 900 pb
|align=center| 900 pb
 +
|align=center| 1 & 2
 +
|align=center| 11-12 & 2-3
|-
|-
! rowspan="2" style="background: #ccccff;" | D103
! rowspan="2" style="background: #ccccff;" | D103
Line 344: Line 180:
! rowspan="2"| 2500 pb
! rowspan="2"| 2500 pb
! rowspan="2"| -
! rowspan="2"| -
 +
|align=center| 2
 +
|align=center| 4-5
|-
|-
|align=center|2
|align=center|2
-
|align=center|
+
|align=center| 2
-
|align=center|
+
|align=center| 6-7
|-
|-
! rowspan="2" style="background: #ccccff;" | D104
! rowspan="2" style="background: #ccccff;" | D104
Line 359: Line 197:
! rowspan="2"| 2500 pb
! rowspan="2"| 2500 pb
! rowspan="2"| -
! rowspan="2"| -
 +
|align=center| 2
 +
|align=center| 8-9
|-
|-
|align=center|2
|align=center|2
-
|align=center|
+
|align=center| 2
-
|align=center|
+
|align=center| 10-11
|-
|-
! rowspan="4" style="background: #ccccff;" | D105
! rowspan="4" style="background: #ccccff;" | D105
Line 374: Line 214:
|align=center| 3000 pb  
|align=center| 3000 pb  
|align=center| -
|align=center| -
 +
|align=center| 2 & 3
 +
|align=center| 12 & 2
|-
|-
|align=center|2
|align=center|2
! rowspan="2"| XbaI
! rowspan="2"| XbaI
! rowspan="2"| PstI
! rowspan="2"| PstI
-
! rowspan="2"|  
+
! rowspan="2"| BI
! rowspan="2"| 2057 pb
! rowspan="2"| 2057 pb
! rowspan="2"| 707 pb
! rowspan="2"| 707 pb
! rowspan="2"| 2000 pb  
! rowspan="2"| 2000 pb  
! rowspan="2"| 700 pb
! rowspan="2"| 700 pb
 +
|align=center| 3
 +
|align=center| 3-4
|-
|-
|align=center|3
|align=center|3
-
|align=center|
+
|align=center| 3
-
|align=center|
+
|align=center| 5-6
|-
|-
|align=center|4
|align=center|4
| align=center|EcoRI
| align=center|EcoRI
| align=center|SpeI
| align=center|SpeI
-
| align=center|
+
| align=center|FI
| align=center| 2056 pb
| align=center| 2056 pb
| align=center| 708 pb
| align=center| 708 pb
|align=center| 2000 pb
|align=center| 2000 pb
|align=center| 700 pb
|align=center| 700 pb
 +
|align=center| 3
 +
|align=center| 7-8
|-
|-
! rowspan="4" style="background: #ccccff;" | D106
! rowspan="4" style="background: #ccccff;" | D106
Line 407: Line 253:
|align=center| 3000 pb
|align=center| 3000 pb
|align=center| -
|align=center| -
 +
|align=center| 3
 +
|align=center| 9-10
|-
|-
|align=center|2
|align=center|2
! rowspan="2"| XbaI
! rowspan="2"| XbaI
! rowspan="2"| PstI
! rowspan="2"| PstI
-
! rowspan="2"|  
+
! rowspan="2"| BI
! rowspan="2"| 2057 pb
! rowspan="2"| 2057 pb
! rowspan="2"| 725 pb
! rowspan="2"| 725 pb
! rowspan="2"| 2000 pb
! rowspan="2"| 2000 pb
! rowspan="2"| 700 pb
! rowspan="2"| 700 pb
 +
|align=center| 3
 +
|align=center| 11-12
|-
|-
|align=center|3
|align=center|3
-
|align=center|
+
|align=center| 4
-
|align=center|
+
|align=center| 2-3
|-
|-
|align=center|4
|align=center|4
| align=center|EcoRI
| align=center|EcoRI
| align=center|SpeI
| align=center|SpeI
-
| align=center|
+
| align=center|FI
| align=center| 2756 pb
| align=center| 2756 pb
| align=center| 726 pb
| align=center| 726 pb
|align=center|  2500 pb
|align=center|  2500 pb
|align=center| 700 pb
|align=center| 700 pb
 +
|align=center| 4
 +
|align=center| 4-5
|-
|-
! rowspan="2" style="background: #ccccff;" |D107
! rowspan="2" style="background: #ccccff;" |D107
Line 440: Line 292:
|align=center| 6000 pb
|align=center| 6000 pb
|align=center| 1000 pb
|align=center| 1000 pb
 +
|align=center| 4
 +
|align=center| 6-7
|-
|-
|align=center| 2
|align=center| 2
Line 449: Line 303:
|align=center| 5000 pb
|align=center| 5000 pb
|align=center| 100 pb
|align=center| 100 pb
 +
|align=center| 6
 +
|align=center| 9
|-
|-
! rowspan="2" style="background: #ccccff;"|D108
! rowspan="2" style="background: #ccccff;"|D108
Line 460: Line 316:
|align=center| 2500 pb
|align=center| 2500 pb
|align=center| 1000 pb
|align=center| 1000 pb
 +
|align=center| 4
 +
|align=center| 8-9
|-
|-
|align=center| 2
|align=center| 2
Line 469: Line 327:
|align=center| 2500 pb
|align=center| 2500 pb
|align=center| 1000 pb
|align=center| 1000 pb
 +
|align=center| 6
 +
|align=center| 10
|-
|-
! rowspan="2" style="background: #ccccff;"|D109
! rowspan="2" style="background: #ccccff;"|D109
Line 480: Line 340:
|align=center| 4000 pb
|align=center| 4000 pb
|align=center| 1000 pb
|align=center| 1000 pb
 +
|align=center| 4
 +
|align=center| 10-11
|-
|-
|align=center| 2
|align=center| 2
Line 489: Line 351:
|align=center| 3000 pb
|align=center| 3000 pb
|align=center| 1000 pb
|align=center| 1000 pb
 +
|align=center| 6
 +
|align=center| 11
|-
|-
! rowspan="2" style="background: #ccccff;"|D110
! rowspan="2" style="background: #ccccff;"|D110
Line 500: Line 364:
|align=center| 2000 pb
|align=center| 2000 pb
|align=center| 800 pb
|align=center| 800 pb
 +
|align=center| 4 & 5
 +
|align=center| 12 & 2
|-
|-
|align=center| 2
|align=center| 2
Line 509: Line 375:
|align=center| 2500 pb
|align=center| 2500 pb
|align=center| 1000 pb
|align=center| 1000 pb
 +
|align=center| 6
 +
|align=center| 12
|-
|-
! rowspan="2" style="background: #ccccff;"|D111
! rowspan="2" style="background: #ccccff;"|D111
Line 520: Line 388:
|align=center| 4000 pb
|align=center| 4000 pb
|align=center| 600 pb
|align=center| 600 pb
 +
|align=center| 5
 +
|align=center| 3-4
|-
|-
|align=center| 2
|align=center| 2
Line 529: Line 399:
|align=center| 4500 pb
|align=center| 4500 pb
|align=center| 700 pb
|align=center| 700 pb
 +
|align=center| 8
 +
|align=center| 2-3
|-
|-
! rowspan="1" style="background: #ccccff;"|D116
! rowspan="1" style="background: #ccccff;"|D116
Line 538: Line 410:
! rowspan="1"| 2100 pb
! rowspan="1"| 2100 pb
! rowspan="1"| 883 pb
! rowspan="1"| 883 pb
-
|align=center|
+
! colspan="4"| Not realised
|align=center|
|align=center|
|-
|-
Line 551: Line 423:
|align=center| 2000 pb
|align=center| 2000 pb
|align=center| 800 pb
|align=center| 800 pb
 +
|align=center| 5
 +
|align=center| 5-6
|-
|-
! rowspan="1" style="background: #ccccff;"|D118
! rowspan="1" style="background: #ccccff;"|D118
Line 562: Line 436:
|align=center| 3000 pb
|align=center| 3000 pb
|align=center| -
|align=center| -
 +
|align=center| 5
 +
|align=center| 7-8
|-
|-
! rowspan="1" style="background: #ccccff;"|D119
! rowspan="1" style="background: #ccccff;"|D119
Line 573: Line 449:
|align=center| 6000 - 3000 pb
|align=center| 6000 - 3000 pb
|align=center| -
|align=center| -
 +
|align=center| 5
 +
|align=center| 9-10
|-
|-
! rowspan="5" style="background: #ccccff;" | D120
! rowspan="5" style="background: #ccccff;" | D120
Line 582: Line 460:
! rowspan="2"| 2057 pb
! rowspan="2"| 2057 pb
! rowspan="2"| 37 pb
! rowspan="2"| 37 pb
-
|align=center| Not digested
+
! rowspan="2"| not digested
-
|align=center| Not digested
+
! rowspan="2"| not digested
 +
|align=center| 7
 +
|align=center| 2
|-
|-
|align=center|2
|align=center|2
-
|align=center|
+
|align=center| 7
-
|align=center|
+
|align=center| 5-6
|-
|-
|align=center|3
|align=center|3
Line 597: Line 477:
|align=center| 1600 pb
|align=center| 1600 pb
|align=center| -
|align=center| -
 +
|align=center| 6
 +
|align=center| 2
|-
|-
|align=center|4
|align=center|4
Line 604: Line 486:
! rowspan="2"| 2080 pb
! rowspan="2"| 2080 pb
! rowspan="2"| 14 pb
! rowspan="2"| 14 pb
-
|align=center| 3000 - 2000 pb
+
! rowspan="2"| 3000 - 2000 pb
-
|align=center| -
+
! rowspan="2"| -
 +
|align=center| 5
 +
|align=center| 11
|-
|-
|align=center|5
|align=center|5
-
|align=center|
+
|align=center| 5
-
|align=center|
+
|align=center| 12
|-
|-
! rowspan="1" style="background: #ccccff;"|D121
! rowspan="1" style="background: #ccccff;"|D121
! rowspan="1" style="background: #ccccff;"|E0422
! rowspan="1" style="background: #ccccff;"|E0422
-
|1
+
|align=center|1
-
|XbaI
+
|align=center|XbaI
-
|PstI
+
|align=center|PstI
-
| FV
+
|align=center| FV
-
| 2057 pb
+
|align=center| 2057 pb
-
| 939 pb
+
|align=center| 939 pb
-
| 3000 pb
+
|align=center| 3000 pb
-
| Not digested
+
|align=center| Not digested
 +
|align=center| 6
 +
|align=center| 3-5
|-
|-
! rowspan="1" style="background: #ccccff;"|D122
! rowspan="1" style="background: #ccccff;"|D122
! rowspan="1" style="background: #ccccff;"|E0840
! rowspan="1" style="background: #ccccff;"|E0840
-
|1
+
|align=center|1
-
|XbaI
+
|align=center|XbaI
-
|PstI
+
|align=center|PstI
-
| FV
+
|align=center| FV
-
| 2057 pb
+
|align=center| 2057 pb
-
| 900 pb
+
|align=center| 900 pb
-
| 2500 pb
+
|align=center| 2500 pb
-
| Not digested
+
|align=center| Not digested
-
 
+
|align=center| 6
 +
|align=center| 6-8
|}
|}
-
gel1<br>
+
 
-
[[Image:Gel1.jpg| gel1]]<br>
+
 
-
gel2<br>
+
==> Conclusion: Most of the digestion have succeed
-
[[Image:Gel2.jpg| gel2]]<br>
+
 
-
gel3<br>
+
== Extraction of the DNA  ==
-
[[Image:Gel3.jpg| gel3]]<br>
+
 
-
gel4<br>
+
 
-
[[Image:Gel4.jpg| gel4]]<br>
+
* Cutting of the parts of interest, for all the digestion that have migrated on the gels
-
gel5<br>
+
* Store of all the piece of gel O/N at -20°C.
-
[[Image:Gel_5bis.jpg‎| gel5]]<br>
+
-
gel6<br>
+
-
[[Image:Gel_6bis.jpg‎| gel5]]<br>
+
-
gel7<br>
+
-
[[Image:Gel_7bis.jpg‎| gel5]]<br>
+
-
gel8<br>
+
-
[[Image:Gel_8bis.jpg‎| gel5]]<br>
+

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.
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