12.Phase DNA の精製 -リキッド・ライセート法- （横溝岳彦）
Version up @1998年3月18日
難しいといわれるリキッド・ライセート法だがやってみたら簡単で、大量かつきれいなファージが取れました。
Phage dilution buffer;20 mM Tris, 20 mM MgCl2, pH 7.5
NZYM medium (For 1 litter):NZ amine 10 g, yeast extract 5 g, NaCl 5 g, MgSO4-7H2O 2 g, pH 7.5
1)Culture Host cells in 200 ml NZYM medium to O.D.600=0.1 at 37 degree in 1 L flask.
2)Add phage solution (high titer is better, at least 10e8)
3)Continue culture overnight
4)add 5 ml chloroform, culture for 10 min.
5)5000 x g for 10 min. Take sup. (Sedimentation of E. coli, and debris)
6)Add 8 g NaCl, 20 g PEG powder (dissolve completely!), on ice 3 hrs.
7)5000 x g for 30 min. discard sup. Leave on paper towel (inverted) for 5 min.
8)Suspend ppt in 7 ml Phage dilution buffer. Transfer to Falcon 50 ml tube.
9)Recover residual phage in 3 ml Phage dilution buffer, and conbine to 8).
10)10,000 x g for 10 min, Take sup (sedimentation of insoluble materials)
11)Add DNase I (20 mg/ml) 10 micro L, and RNase (10 mg/ml) 10 micro L, and incubate at 37 degree for 30 min. (disruption of E-coli derived DNA and RNA)
12)Add 0.75 ml 0.5 M EDTA-Na2, R.T 5 min, and 65 degree for 30 min. (Inactivation of DNase)
13)Add 0.5 ml 10 % SDS, 65 degree for 30 min. (Disruption of Phage particule)
14)Phenol extraction ~2 times.
15)Phe/Chl extraction ~2-4 times. (Until no debri observed)
16)Chl extraction once to remove Phenol.
17)EtOH or Isopropanol precipitation.