Editing Competent-Cells (section)

== CTAB Method for electroporation ==

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287956/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287956/]

Grow E. coli (BL21DE3, XL1blue, and DH5-α), Bifidobacterium sp., and K. lactis in their respective media to (OD600 0.4–0.6) an exponential phase.

Spin down cells at 6000 ×g for 10 min at 4 °C

Resuspended at one tenth of their original volume in ice-cold 1 mM HEPES buffer.

Wash again and spin

Resuspend cells in 1 mM HEPES buffer containing 1 μg/ml&nbsp; (1mg/1000 ml) CTAB.

1 mg/1000 ml CTAB: dilute from 100X (100 &nbsp;μg/ml, 50 mg CTAB-->500 ml H2O) stock, add&nbsp;5&nbsp;ul of 100&nbsp; μg/ml&nbsp; stock to 500 ml autoclaved H2O

Incubate for 15 min on ice.

Wash cells with water twice.

Resuspend cells in cold 10% glycerol solution.

Aliquot 100 μl cells

Store at -80 oC

Incubated at 4 °C for 5 min. The transformation mix was transferred to electroporation cuvettes (Bio-Rad, Hercules, and CA), and cells were electroporated in a Bio-Rad Gene Pulsar according to the manufacturer's directions for transformation of bacteria, and yeasts. The following fixed parameters of the Bio-Rad Gene Pulsar were followed, for electroporation of E. coli 0.2 cm cuvette, resistance 25 μF, and 200 V, bifidobacteria 0.2 cm cuvette, 25 μF resistance and 200 V, and yeast 0.2 cm cuvette, 25 μF resistance and 200 V. Subsequently, 0.9 ml of fresh medium was added, and the cells were grown at 37 °C. E. coli cells with shaking at 200 rpm for 1 h, Bifidobacterium sp., and K. lactis, without shaking were incubated.