Team:Paris/July 26

From 2008.igem.org

(Difference between revisions)
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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"  
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===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 ===
-
Gel 1, 2, 3, 4, 5 = 0.8%   et    Gel 6 = 1,5%
+
Gel 1, 2, 3, 4, 5 = '''0.8%'''    et    Gel 6 = '''1,5%'''
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! rowspan="1"| 3
! rowspan="1"| 3
|}
|}
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 +
 +
==> conclusion : all the digestion have succeed.....GREAT !
 +
 +
 +
 +
== Extraction of the DNA  ==
 +
 +
 +
* 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.

Revision as of 10:41, 28 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 [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

Gel 1, 2, 3, 4, 5 = 0.8% et 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
3 EcoRI XbaI FV 2076 pb 15 pb 2000 pb - 1 2
4 SpeI PstI BV 2077 pb 14 pb 2000 pb - 1 3
5 1 4
D101 J23101 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 700 pb 1 5
2 1 6
D102 J23109 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 700 pb 1 7
2 1 8
D103 R0079 1 SpeI PstI BV 2222 pb 14 pb 2000 pb - 1 9
2 1 10
D104 R0040 1 SpeI PstI BV 2119 pb 14 pb 2000 pb - 1 11
2 1 12
D105 S03154 1 SpeI PstI BV 2750 pb 14 pb 3000 pb - 2 5
2 XbaI PstI BI 2057 pb 707 pb 2000 pb 700 pb 2 2
3 2 3
4 EcoRI SpeI FI 2056 pb 708 pb 2000 pb 700 pb 2 4
D106 S03879 1 SpeI PstI BV 2768 pb 14 pb 2700 pb - 2 6
2 XbaI PstI BI 2057 pb 725 pb 2000 pb 700 pb 2 7
3 2 8
4 EcoRI SpeI FI 2756 pb 726 pb 3000 pb 700 pb 2 9
D107 C0079 1 EcoRI SpeI FI 4402 pb 779 pb 5000 pb 800 pb 2 10
2 XbaI PstI BI 4003 pb 778 pb 5000 pb 800 pb 2 11
D108 C0179 1 EcoRI SpeI FI 4402 pb 746 pb 2000 pb 800 pb 2 12
2 XbaI PstI BI 4403 pb 745 pb 2000 pb 800 pb 3 2
D109 E0030 1 EcoRI SpeI FI 3166 pb 746 pb 3000 pb 700 pb 3 3
2 XbaI PstI BI 3167 pb 745 pb 3000 pb 700 pb 3 4
D110 E0040 1 EcoRI SpeI FI 2056 pb 743 pb 2000 pb 700 pb 3 5
2 XbaI PstI BI 2057 pb 742 pb 2000 pb 700 pb 3 6
D111 E1010 1 EcoRI SpeI FI 4402 pb 704 pb 5000 pb 700 pb 3 7
2 XbaI PstI BI 4403 pb 703 pb 5000 pb 700 pb 3 8
D116 J23100 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 900 pb 3 9
2 3 10
D117 J23107 1 SpeI PstI BV 2100 pb 883 pb 2000 pb 900 pb 3 11
2 3 12
D118 B0015 1 EcoRI XbaI FV 3303 pb 15 pb 3000 pb - 4 2
2 4 3
D119 I0500 1 SpeI PstI FV 5621 pb 14 pb 6000 pb - 4 4
2 4 5
D120 B0030 1 XbaI PstI BI 2057 pb 37 pb 2000 pb 50 pb 6 4
2 6 5
3 EcoRI XbaI FV 2079 pb 15 pb 2200 pb - 4 6
4 SpeI PstI BV 2080 pb 14 pb 2000 pb - 4 7
5 4 8
D121 E0422 1 XbaI PstI BI 2057 pb 939 pb 2000 pb 1100 pb 4 9
2 4 10
3 4 11
D122 E0 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 !


Extraction of the DNA

  • 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.

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