Maloof Lab:Cell Transformation

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E. coli (DH5α chemically-competent) Transformation Thaw one aliquot of DH5α competent cells (from –80°C freezer) on ice Add 1-5 μl DNA and 75 μl competent cells to new cell culture tube. Do not mix the cells by pipetting. Just tap the tube gently Incubate on ice for 30 minutes Heat shock in 42°C water bath for exactly 30 seconds. Do not mix or shake Cool on ice Add 900 μl liquid media (e.g. LB) to cell culture tube Shake at 37°C for 40-60 minutes Centrifuge at 5000 rpm for 2 minutes Remove all but 100-150 μl of the LB and resuspend pellet Spread 100-150 μl on plate of media + antibiotic (e.g. LB agar + carb) Incubate plates up-side-down, overnight at 37°C