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Revision as of 20:44, 16 October 2016

Protocol 6 : E.Coli Dh5α and BL21 transformation (Heat-shock)

Aim: Introduce plasmid DNA in E.Coli bacteria

Thaw a tube of DH5-alpha competent cells on ice for 10 min.
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.