Journal article Open Access

Transformation of plasmid DNA to competent E. Coli cells

sprotocols

Authors: Chenzhong Kuang ### Material and Reagents 1. SOC - 2% Tryptone - 0.5% Yeast Extract - 10mM NaCl - 10mM MgSO4 - 10mM MgCl2 - 1.5 mL microfuge tubes - 42° C waterbath - Ice - 37° C shaker ### Protocol 1. Thaw competent cells on ice. 20–200µL per tube - Add max. 20µL of a ligation reaction - Mix very gently! - Incubate the tubes on ice for 30 min - Heat shock the cells for 45 sec to 2 min at 42°C - Place the tubes immediately on ice for at least 2 min - Add 800µL of SOC medium to each tube - Incubate for 1 hour at 37°C and shake vigorously - Spin down briefly and remove most supernatants - Resuspend cell pellet with the rest SOC medium in the tube by pipetting - Plate out the suspension on a LB agar plate containing the appropriate antibiotic. - Incubate the plates overnight at 37°C ### Remarks 1. I have use this protocol to transform DH5 alpha competent cells successfully - When transforming purified plasmid into competent cells, I just add 1µL plasmid DNA solution. Then plate out only 10–20µL bacterial suspension to the plate instead of all - Efficiency depends on ligation reaction and competent activity.

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