细菌转化方法步骤(英文版)_细菌转化步骤

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Using ice-cold CaCl2 to prepare competent cell, protocol 1）Incubate the single colony from the LB solid selection plate in LB liquid medium at the 37℃ for 16-20h,then pipette at the 1:10 dilution to the fresh LB liquid medium ,incubate to the OD A600 about 0.5 at the 37℃(for 2-3h)..2）Put the flask on the ice for 10~15min ,then collect the cells at the 3000g for 5min in 4℃,put the 0.1 M CaCl2 on the ice before.3）Discard the total supernatant as poible as you can ,Add the ice-cold CaCl2 to the cell mixture in 1/3 volume of the.medium, carefully flick the tube 4–5 times to mix cells,then put on the ice for 15-20min.4）Centrifuge at the 3000g for 5min in 4℃, repeat the 3)5)Discard the total supernatant, add the ice-cold CaCl2 to the cell mixture in 1/25 volume of the.medium, carefully flick to mix cells.6）Keep the competent cells in-80℃ with adding the15%-20% glycerol.Transformation Protocol 1.Thaw a tube of Competent cells on ice until the last crystals disappear(about 10 min).Mix gently and carefully pipette 50 μl of cells into a transformation tube on ice.2.Add 1–5 μl containing 1 pg–100 ng of plasmid DNA to the cell mixture.Carefully flick the tube 4–5 times to mix cells and DNA.Do not vortex.3.Place the mixture on ice for 30 minutes.Do not mix.4.Heat shock at exactly 42°C for exactly 42seconds.Do not mix.5.Place on ice for 3 minutes.Do not mix.6.Pipette 800 μl of room temperature LB into the mixture.7.Place at 37°C for 60 minutes.Shake vigorously(180 rpm)or rotate.8.Warm selection plates to 37°C.9.Mix the cells thoroughly by flicking the tube and inverting, then centrifuge at 4000 rpm for 1 min,then discard the supernatant to keep 300ul in the tube.10.Spread 100 μl onto a selection plate and incubate overnight at 37°C.