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Team:Macquarie Australia/WetLab/Protocols/Ligation
From 2014.igem.org
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<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/SDSPAGE">SDS-PAGE</a></li> | <li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/SDSPAGE">SDS-PAGE</a></li> | ||
<li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Heat">Heat Shock</a></li> | <li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Heat">Heat Shock</a></li> | ||
| + | <li><a href="https://2014.igem.org/Team:Macquarie_Australia/WetLab/Protocols/Operons">Induction of Operons</a></li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
Latest revision as of 13:09, 17 October 2014
Composite Part Ligation
The assembly of composite parts from two existing BioBricks i.e. BioBrick plasmid A (parent vector) and BioBrick plasmid B (gene to be inserted) was performed through a restriction digest/ligation protocol.
- 200ng of each BioBrick plasmid was digested as follows: Plasmid BioBrick A with NEB restriction enzymes SpeI and PstI; plasmid BioBrick B with XbaI and PstI, according to supplier protocol. (1hr @37oC, then 20mins @80oC).
- 1µL of Fast alkaline phosphatase (Thermo Scientific) was added to reaction tube of plasmid BioBrick A to dephosphorylate the gene fragments and prevent re-ligation of the parent vector. Reaction tubes were incubated at 37oC for 60 mins, followed by a deactivation step at 80oC for 20 mins.
- Ligation was then performed with an insert to vector molar ratio of 3:1. 1µL of T4 ligase (NEB) was added to the mix for ligation, according to supplier protocol. Ligation reactions were performed at 37oC for 60 mins and kept on ice for transformation into chemical competent cells.
