Transformation of Escherichia coli cells Sample Clauses

Transformation of Escherichia coli cells. NEB®-10 beta E.coli cells normally stored at -80°C were thawed on ice. 1 μl of the LR reaction mix or plasmid DNA was then added to the cells, without vigorous pipetting. The mixtures were then put on ice for 30 minutes. Bacteria were then transformed by heat shock. Tubes containing the bacteria and DNA were incubated at 42°C for 30 seconds and were then returned to ice for another 2 minutes. 500 μl of LB-broth were added to the transformed E.coli cells which were then shaken at 37°C for 1 hour at 225rpm. 100μl of the transformed bacteria were then spread onto LB-agar plates containing the appropriate antibiotic. Plates were then placed inverted in an incubator set at 37°C overnight. On the following day 3 colonies were picked and grown in LB broth supplemented with the appropriate selection antibiotic. A small fraction of the yielded culture was used to make a glycerol stock while the rest was used for DNA purification.
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