This method is suitable for the extraction of small amount of plasmid DNA, which can be directly used for PCR amplification.
1.Take 1.5ml of bacterial culture in EP tube, centrifugate at 4000rpm for 1min, discard supernatant, and make bacterial precipitation as dry as possible.
2.Resuspend bacterial precipitation in 100 μ l solution I precooled with ice(50 mmol / L glucose,10 mmol/LEDTA pH 8.0，25 mmol/L Tris-HCl pH 8.0).Severe oscillation.
3.Add 200 μ l of newly prepared solution II(0.2 mol/L NaOH，1％SDS（m/v）),Close the EP pipe orifice, quickly reverse the centrifuge tube 5 times to mix the mixture, ensure that the entire inner surface of the centrifuge tube contacts solution II, do not vortex, and put it in the ice bath.
4.Add 150 μ l precooling solution III(60 ml of 5 mol / L potassium acetate, 11.5 ml of glacial acetic acid, 28.5 ml of H2O per 100 ml of solution III),close the EP pipe orifice, turn it upside down several times, make solution III disperse evenly in the viscous bacterial lysate, and then place the pipe on ice for 3-5 minutes.
5.Centrifugation at maximum speed for 5min, take the supernatant and put it into another new EP tube.
6.Double stranded DNA was precipitated with twice the volume of ethanol at room temperature, mixed with vibration and placed at room temperature for 2 minutes, centrifuged at maximum speed for 5 minutes.
7.Carefully remove the supernatant and invert the centrifuge tube onto the filter paper so that all the liquid flows out,then remove all the droplets attached to the pipe wall.
8.Add 1ml 70% ethanol to wash the precipitate, shake and mix, centrifugate with 12000g for 2 minutes, discard the supernatant, place the open EP tube at room temperature to volatilize the ethanol until there is no visible liquid in the EP tube(5~10 minutes).Dissolve with appropriate amount of ddH2O.
9.Incubate with 0.5 μ l RNase at 37 ℃ for 5-10 minutes.
10.Electrophoretic identification.