Cell ( E. coli ) down (maximum speed for 30 seconds)
3
Cell (E. coli) down (maximum speed for 30 seconds) rnatant (medium) bacterial pellet Alkaline lysis solution I (100 µl)-vigorously vortexing Alkaline lysis solution II (200 µl)-inverting five times incubate on Alkaline lysis solution III (150 µl)-inverting several times incubate on Centrifuge 12000 rpm for 10 min at 4 o C. supernatant (plasmids, proteins, and RNAs) hromosomes) Add an equal volume of phenol:chloroform (400 µl) Transfer the supernatant to a new tube.
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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 - PowerPoint PPT Presentation
Transcript of Cell ( E. coli ) down (maximum speed for 30 seconds)
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)
Add an equal volume of phenol:chloroform (400 µl)
Transfer the supernatant to a new tube.
Aqueous upper layer (plasmids and RNAs)
Phenol:chloroform (proteins)
maximum speed for 2 min
*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)
Transfer the 400 µl of aqueous upper layer to a new tube
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
