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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

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slide1

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)

slide2

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)

slide3

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