Team:Evry/Notebook/Transposons/14.10.14

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Digestion of BBa_1413044 by BglII :
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Digestion of BBa_K1413001 by E/S :
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<br>50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (20 ng/ul) BBa_1413044 in pSB1C3, 1 ul BglII, 39 ul H2O.
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<br>50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (35 ng/ul) BBa_1413001 in pSB1C3, 1 ul E/S, 39 ul H2O. 1h
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<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
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Gel electrophoresis:
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GEEEEEEEEEEEEEEEEEEEEEELLLLLLLLLL
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<img src="" width="50%"/>
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Purification sur gel:
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Digestion of Kan 82/83 by E/S & X/P:
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<br>50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul (30 ng/ul) Kan 82/83 in pSB1C3, 1 ul E/S or X/P, 398ul H2O. 1h
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<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
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Ligation:
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I° strategy:
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<br> 20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 3 ul Kan 82/83 + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1h
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II° Strategy:
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<br> 20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1h
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 +
Clean up:
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II° strategy:
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Digestion: II° strategy
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<br> 50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul Kan 82/83 in pSB1C3, 1 ul E/S or X/P, 398ul H2O. 1h
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Kit thermoscientific "Gel extraction". Elution final in 30 ul.
Kit thermoscientific "Gel extraction". Elution final in 30 ul.
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Revision as of 20:14, 17 October 2014

Picture

== Ligation of BBa_K1413001==
strategy:

Digestion of BBa_K1413001 by E/S :
50 ul final volume, 5 ul Buffer NeB 3.1, 5 ul (35 ng/ul) BBa_1413001 in pSB1C3, 1 ul E/S, 39 ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
Digestion of Kan 82/83 by E/S & X/P:
50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul (30 ng/ul) Kan 82/83 in pSB1C3, 1 ul E/S or X/P, 398ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution Ligation: I° strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 3 ul Kan 82/83 + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1h II° Strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul digestion product BBa_K1413001 by E/S + 5 BBa_1413044 transposon plasmid. 4 ul H2O. 1h Clean up: II° strategy: Digestion: II° strategy
50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul Kan 82/83 in pSB1C3, 1 ul E/S or X/P, 398ul H2O. 1h Kit thermoscientific "Gel extraction". Elution final in 30 ul.
== Obtaining the Kan gene on pNK2 ==
PCR: enzyme: Q5; template: 150 ng pNK2; oligo: F 82/ R 83; Tm tested: 55/60/65; elongation time: 1m30s

Purification sur gel:
Kit thermoscientific "Gel extraction". Elution final in 30 ul.

Oct 13

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