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Team:Evry/Notebook/Transposons/14.10.14
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Latest revision as of 22:55, 17 October 2014
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, 38ul 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. 1 ul T4 ligase. 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.1 ul T4 ligase. 1h
Clean up:
II° strategy:
Ligation product by Thermoscientific "PCR clean up" Kit in 30 ul final elution
Digestion: II° strategy
50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul ligation product, 1 ul E/X, 39 ul H2O. 1h
Then Thermoscientific "PCR clean up" Kit in 30 ul final elution
Ligation:
II° Strategy:
20 ul final volume, 5 ul Buffer T4 fermentas, 3 ul ligation product E/X + 5 ul Kan 82/83 E/S. 4 ul H2O. 1h
Transformation: electrocompetent
50ul electrocompetent cells
2 ul Ligation product of I° strategy / II° strategy in E.coli DH5alpha.
