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Team:Evry/Notebook/Transposons/14.10.14
From 2014.igem.org
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Digestion of Kan 82/83 by E/S & X/P: | Digestion of Kan 82/83 by E/S & X/P: | ||
| - | <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, | + | <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, 38ul H2O. 1h |
<br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution | <br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution | ||
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II° Strategy: | II° Strategy: | ||
<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 | <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 | ||
| + | |||
Clean up: | Clean up: | ||
II° strategy: | II° strategy: | ||
| + | Ligation product by Thermoscientific "PCR clean up" Kit in 30 ul final elution | ||
| + | |||
Digestion: II° strategy | Digestion: II° strategy | ||
| - | <br> 50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul | + | <br> 50 ul final volume, 5 ul Buffer NeB 4.1, 5 ul ligation product, 1 ul E/X, 39 ul H2O. 1h |
| + | <br>Then Thermoscientific "PCR clean up" Kit in 30 ul final elution | ||
| + | |||
| + | Ligation: | ||
| + | |||
| + | II° Strategy: | ||
| + | <br> 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: | ||
| + | |||
| + | |||
| + | |||
| - | |||
| - | |||
== Obtaining the Kan gene on pNK2 == | == Obtaining the Kan gene on pNK2 == | ||
<br> | <br> | ||
Revision as of 20:19, 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. 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:
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:
== 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.
