Difference between revisions of "Team:Ionis Paris/Protocol 6"
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<h4 class="sidebar_Hd">Sources</h4> | <h4 class="sidebar_Hd">Sources</h4> | ||
| − | <p><li><a href="https://www.neb.com/protocols/2012/05/21/transformation-protocol">NEB Transformation Protocol</a></li></p> | + | <p><li><a href="https://www.neb.com/protocols/2012/05/21/transformation-protocol"><font color="DeepPink">NEB Transformation Protocol</font></a></li></p> |
| − | <p><li><a href="https://www.addgene.org/plasmid-protocols/bacterial-transformation/">Addgene Bacterial Transformation protocol</a></li></p> | + | <p><li><a href="https://www.addgene.org/plasmid-protocols/bacterial-transformation/"><font color="DeepPink">Addgene Bacterial Transformation protocol</font></a></li></p> |
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Revision as of 15:06, 18 October 2016
Thaw a tube of DH5-alpha competent cells on ice for 10 min.
Protocol 6 : E.Coli Dh5α and BL21 transformation (Heat-shock)
Aim: Introduce plasmid DNA in E.Coli bacteria
Mix gently and carefully pipette 50 µL of cells into a transformation tube on ice
Add 1-5µL plasmid DNA to the cell mixture (one tube for each ratio)
Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 min. Do not mix.
Heat shock at exactly 42°C for 45 s. Do not mix.
Place on ice for 5 min. Do not mix.
Pipette 250µL of room temperature SOC into the mixture.
Place at 37°C for 60 min at 250 rpm.
Warm selection plates to 25°C.
Mix the cells by flicking the tube and inverting.
Spread 100µL of cells onto selection plates.
Incubate all the plates O/N at 37°C.
