11.Phase DNA の精製 －プレート・ライセート法－ （横溝岳彦）
Version up @1998年3月18日
Phage dilution buffer;20 mM Tris, 20 mM MgCl2, pH 7.5
1)Culture phage in 15 x 9 cm plate for O/N.
2)Add 8 ml phage dilution buffer and shake for 3 h at R.T.
3)Recover the dilution buffer and wash plates again with 3 ml buffer.
4)8500 rpm for 10 min. Take sup. (Sedimentation of E. coli, and debris)
5)Add DNAse I (20 micro g/micro l) 1 micro l, and RNAse (10 micro g/micro l) 1 micro l, incubate at 37 degree for 10 min. (Disruption of E. coli-derived DNA and RNA)
6)Add same vol. of PEG sol., incubate on ice for 2 h. (Sedimentation of phage particle)
7)8500 rpm for 15 min. Disgard sup. and stand inverted on paper towels.
8)Suspend in 700 micro l Phage dilution buffer
9)10,000 x g for 10min, Take sup. (Removal of insolubles)
10)Add 50 micro l 0.5 M EDTA-Na2, R.T. 5 min, and then, 65 degree for 30 min. (Inactivation of DNAse)
11)Add 30 micro l 10% SDS, 65 degree for 30 min. (Disruption of phage particle)
12)Phenol extraction x 2 (Removal of protein and etc.)
13)Phe/Chl extraction x 2 (Times should be changed by the amount of the debris)
14)Chl extraction x 1
15)Add same vol of 7.5 M NH3oAc, on ice 30 min. (Removal of contaminated agarose)
16)10,000 x g for 10min, Take sup.
17)EtOH precipitaion.