Method
- Dilute a fresh culture of host cells (e.g., JM101) 1:20
in 2 ml of L broth and infect with phage from a purified M13 plaque.
- Incubated at 37°C with shaking for 6-8 hours; transfer 1 ml
culture to eppendorf tube and centrifuge for 30 minutes at room
temperature.
- Carefully remove 700 µl of supernatant to a fresh tube and add 30
µl of PEG/NaCl (20% PEG-8000, 2.5 M NaCl). Allow the phage to
precipitate 20 minutes at room temperatue.
- Centrifuge 20 minutes at room temperature; carefully remove the
supernatant fluid with a pipette and wipe off the inside of the
tube.
- Resuspend the phage in 200 µl of TE buffer and extract with 100
µl of phenol 2 times. (Vortex and spin down to separate the layers.
Then extract 1X with SEVAG (chloroform:isoamyl alcohol, 24:1).
- Remove the top layer to a fresh tube than add 20 µl of 3 M sodium
acetate and 400 µl of ethanol. Chill on dry ice for 30 minutes;
centrifuge 30 minutes to collect the precipitated DNA.
- Wash the pellet with 70% ethanol and air dry.
- Resuspend the DNA in 30 µl of TE buffer. It should contain 0.5-1
µg/µl. (This may be estimated by agarose gel.)
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