Phenol-chloroform lysis method of extracting plasmid DNA.docVIP

 PAGE \* MERGEFORMAT 5 ‘Phenol-chloroform lysis method’ of extracting plasmid DNA Authors: Zhang Linlin, Li Xiang, Zhang Dong, Da-Lin, Li Lei, Wang Xin-yang [Keywords:] plasmid DNA; phenol chloroform; extraction; endonuclease; polymerase chain reaction; electrophoresis [Keywords:] plasmid DNA; phenol chloroform; extraction; endonuclease; polymerase chain reaction; electrophoresis 1 Materials and methods 1.1 Materials containing human coxsackie adenovirus receptor (human Coxsackie and adenovirus receptor, hCAR) gene eukaryotic expression vector pIRES2EGFPhCAR built by the Institute; E.coli DH5a bacteria transformed by the Institute for preservation; endonuclease PstI were purchased from TaKaRa company. LB medium (peptone 10 g, yeast extract 5 g, sodium chloride 10 g). Tris saturated phenol, chloroform, 1:1 volume ratio of preparation of the mixture; anhydrous ethanol; RTE solution: 10 mmol / L TrisHCl (pH 8.0), 1 mmol / L EDTA +20 mg / mL RNase A (without DNase). diameter of 35 mm can be used repeatedly a small needle, and 0.22 μ m membrane filters were purchased from Sino-American Biotechnology biotechnology company. 1.2 Methods ① were picked from the agar plate bacteria transformation positive clones were inoculated into the standard LB medium (containing kanamycin 30 μ g / mL) shaking bacteria 12 h; collection of 1.5 mL bacilli, 8000 g / min centrifugation 3 min , supernatant was precipitated by adding 200 μ L TE, adequate mixing; Add 400 μ L phenol-chloroform (1:1 volume) mixture, intense vibration of 10 s, mixing; 12 000 g / min centrifugation 5 min, 1 mL insulin the needle to collect the supernatant to avoid inhalation of protein deposition layer; supernatant by domestic filter 0.22 μ m filter needle 1; to filter the supernatant added to 2 times the volume of anhydrous ethanol, oscillation 10 s, 12 000 g / min centrifugation 5 min; precipitate dissolved in 20 μ L of the RTE solution, 37 ℃ water bath. ② according to the instructions for PstI en