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Team:Macquarie Australia/WetLab/Protocols/Ligation
From 2014.igem.org
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<h3>Composite Part Ligation</h3> | <h3>Composite Part Ligation</h3> | ||
| - | <p></p> | + | <p> |
| + | 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. | ||
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| + | <ul style="list-style-type: decimal;"> | ||
| + | <li> | ||
| + | 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 @37°C, then 20mins @80°C). | ||
| + | </li> | ||
| + | <li> | ||
| + | 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 37°C for 60 mins, followed by a deactivation step at 80°C for 20 mins. | ||
| + | </li> | ||
| + | <li> | ||
| + | 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 37°C for 60 mins and kept on ice for transformation into chemical competent cells. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | </p> | ||
</div> | </div> | ||
</section> | </section> | ||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 04:48, 16 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 @37°C, then 20mins @80°C).
- 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 37°C for 60 mins, followed by a deactivation step at 80°C 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 37°C for 60 mins and kept on ice for transformation into chemical competent cells.
