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"  
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|-
|-
|align="center"|MP116
|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
|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
|align="center"|MP118
-
|align="center"|B0015
+
|align="center"|[http://partsregistry.org/Part:BBa_B0015 B0015]
-
|align="center"|
+
|align="center"|Double terminator
|-
|-
|align="center"|MP119
|align="center"|MP119
-
|align="center"|I0500
+
|align="center"|[http://partsregistry.org/Part:BBa_I0500 I0500]
-
|align="center"|
+
|align="center"|AraC pBAD
|-
|-
|align="center"|MP120
|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
|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
+
|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>
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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 ===
 +
 +
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%'''
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! 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
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| 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
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! 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
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| 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
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|align=center| 6-8
|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.
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