目的：各種実験に用いるplasmidを大腸菌から調製する
Culture
<from plate , single colony>
↓inoculate to 2 ml of LA-broth / spitz tube
↓incubate at 37℃ with shaking, O / N
prep.
↓on ice 5 min
↓transfer to 1.5 ml tube (decant)
↓cfg. 13,200 rpm, 4℃, 5 min
↓remove sup. (decant)
↓add 100 ul of Lysozime solution
(5 mg of Lysozyme + 1250 ul of 1 × Lys. buffer)
↓vortex
↓RT 5 min
↓add 200 ul of 0.2 N NaOH / 1 % SDS
↓inversion
↓add 150 ul of 3 M KoAc
↓inversion
↓on ice 10 min
↓cfg. 13,200 rpm, 4℃, 15 min
↓upper phase to new tube (decant)
↓add 450 ul of PhOH / CHCl3
↓inversion
↓cfg. 13.200 rpm, RT, 10 min
↓upper phase to new tube (先cut tip)
↓add 400 ul of -20℃ IpOH
↓inversion
↓RT 5 min
↓cfg. 13,200 rpm, 4℃, 10min
↓remove sup. & dry (decant)
↓add 100 ul of RnaseA / TE
(12 ul of RNaseA (10 mg / ml) + 1.2 ml of TE)
↓vortex 5 min
↓incubate at 37℃, 15 min-
↓add 300 ul of KoAc / TE
↓add 400 ul of PhOH / CHCl3
↓vortex 2 min
↓cfg. 13.200 rpm, RT, 10 min
↓upper phase to new tube (先cut tip)
↓add 1 ml of -20℃ 100 % EtOH
↓inversion
↓-80℃, 5 min
↓cfg. 13.200 rpm, 4℃, 15 min
↓70 % EtOH Rinse & dry
↓dissolve in 40 ul of TE
