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This protocol outlines the step-by-step process for isolating plasmids and RNAs from E. coli cells through alkaline lysis. Starting with the preparation of cell supernatant, the procedure involves the sequential addition of alkaline lysis solutions, followed by centrifugation to separate bacterial chromosomes from plasmids and RNAs. The mixture is then treated with phenol:chloroform for further purification. Finally, ethanol precipitation and washing steps are implemented to obtain pure plasmids and RNAs ready for downstream applications.
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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
