10. 2 Phage 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



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.