Plasmid Extraction Protocol from E. coli Using Alkaline Lysis Method
This protocol outlines the alkaline lysis method for efficiently extracting plasmids from E. coli cells. The process begins with cell lysis using a series of alkaline solutions, followed by centrifugation to separate plasmids from cellular debris. The supernatant containing plasmids is treated with phenol:chloroform for further purification, and ethanol precipitation is used to isolate plasmids. The final product is a pellet that can be dissolved in TE buffer with RNase A for downstream applications. This protocol ensures high yields of pure plasmid DNA.
Plasmid Extraction Protocol from E. coli Using Alkaline Lysis Method
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Presentation Transcript
Cell (E. coli) down (maximum speed for 30 seconds) supernatant (medium) bacterial pellet Alkaline lysis solution I (100 µl)-vigorously vortexing Alkaline lysis solution II (200 µl)-inverting five times incubate on R·T for 5 min. Alkaline lysis solution III (150 µl)-inverting several times incubate on ice for 5 min. Centrifuge 12000 rpm for 10 min at 4 oC. pellet supernatant (plasmids, proteins, and RNAs) (bacterial chromosomes) Transfer the supernatant to a new tube. Add an equal volume of phenol:chloroform (400 µl)
Aqueous upper layer (plasmids and RNAs) Phenol:chloroform (proteins) maximum speed for 2 min Transfer the 400 µl of aqueous upper layer to a new tube *Ethanol precipitation Add 0.1 vol Sodium acetate (0.3 M, pH 5.2) of sample volume (40 µl) 2.5 vol 100 % Ethanol of sample volume (1000 µl)
Centrifuge 12000 rpm for 10 min at 4 oC. supernatant (ethanol) pellet (plasmids and RNAs)) Add 70 % ethanol for washing the pellet Spin down (remove the rest of 70 % ethanol.) To eliminate the ethanol perfectly, dry the pellet for 10 min Dissolve the pellet in 30 µl of TE buffer(or ddH2O) containing RNase A
