transformation to E.coli

assays/Mutant Generation/protocols/TransformationBW29427.md

+## Transformation into chemically competent E. coli BW29427 cells
+
+The vector was transformed into 50 μl chemically competent BW29427 *E. coli* cells according to the following heat shock protocol: 2 μl of the vector were gently mixed with 50 μl of competent cells, and the resulting mixture was incubated on ice for 30 min. The mixture was transferred to a water bath at 42 °C for 1 min and put back on ice for 2 min. Then, 1 ml of 50% TSB with 50 μg/ml diaminopimelic acid (DAP; Sigma-Aldrich) was added to the heat-shocked cells, the mixture was left to regenerate at 37 °C for 1 h and then plated on 50% TSA containing 25 μg/ml Kanamycin (Kan) and 50 μg/ml DAP. The plates were incubated at 37 °C overnight. Resulting colonies were validated by colony PCR using the M13F and M13R primers. Colony PCR was performed on at least four separate colonies with 0.4 μl DFS-Taq polymerase (BIORON) in 25 μl reactions containing 2.5 μl 10x incomplete buffer (BIORON), 0.5 10 mM MgC12, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM forward primer, 0.75 μl 10 μM reverse primer, a small fraction of a colony and filled up to 25 μl with nuclease-free water. The tubes were placed in a thermocycler set at the following  program: 94°C for 2 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, then a final extension at 72 °C for 10 min. Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Positive colonies were purified by streaking on new 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml DAP and further verified by Sanger sequencing (Eurofins Scientific) following the manufacturer's protocol.