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
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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)
Transfer the supernatant to a new tube.
Add an equal volume of phenol:chloroform
maximum speed for 2 min
Transfer the 400 µl of aqueous upper layer to a new tube
Add 0.1 vol Sodium acetate (0.3 M, pH 5.2) of sample volume
2.5 vol 100 % Ethanol of sample volume
supernatant (ethanol) pellet (plasmids and RNAs))
Add 70 % ethanol for washing the pellet
(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