Phage DNA isolation

Bob Tacon bob_tcc at yahoo.com
Tue Sep 28 18:50:35 EST 1999


Hello!

A protocol for isolation of lambda phage DNA from phage lysate stated
the following steps (abbreviated):

1. Add 75 ul 0.5 M EDTA to the phage lysate, vortex, add 2 ml phenol,
vortex, add 2 ml chloroform.

2. Centrifuge and remove organic layer.

3. Repeat phenol-chloroform extraction.

4. Extract once with 2 ml chloroform only.

5. Add 0.4 ml of 2 M NaOAc (pH 6.5), vortex, add 10 ml of 95 % EtOH.

6. Centrifuge, discard supernatant, the DNA is contained in the pellet.

I understood that at last what I have in the pellet is the DNA that I
wanted. But I do not know how this exactly works. I know that EtOH is
responsible for precipitating DNA, but what property is it that makes
EtOH precipitate DNA? What is the NaOAc good for in this precipitation.
I guess the other steps have the function of removing non-DNA
substances. How does this work and why does it work so selectively?

Bob Tacon




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