DNA Isolation

We’ll use our favourite E.coli yet again. Poor E.coli!
We take a pellet of it as before, and add T10E1 buffer to it. We mix up the stuff by vortexing.
We won’t do anything as elaborate as what we did while isolating the plasmid DNA- we directly come to THE POINT: 10µl of lysozyme. Using its own enzyme against itself. Sad.
Now that all its membranes are gone, we incubate it for 15 minutes at 370C for the reaction to reach a completion. Then we add 30µl of 10% SDS- that’s for dissolving all the lipid stuff. 3µl of proteinase K takes care of the protein molecules. Just give it some more time: incubate at 370C for half-an-hour.
Next, we shall use a wonderful technique to separate the cell parts- selective solvation. We add 250µl of tris saturated phenol and another 250µl of chloroform. Mix it up by inverting. Follow it up with a little centrifugation and the cell parts all separate out. Chloroform dissolves most of the cell parts while phenol dissolves the protein remains. DNA, as before, remains suspended in water forming hydro-complexes.



Carefully using a micropipette, extract as much of the supernatant as possible (that’s usually 3ml), without disturbing the phenol layer, and transfer it to a new tube. To it, add 150µl of 5M NaCl, and twice its own volume of chilled ethanol. Invert mix and then centrifuge.
A pellet of DNA is formed. Decant the ethanol. This is the familiar ‘salting out’ process that we carried out during our plasmid extraction. This is followed by ‘ethanol wash’ and another centrifugation. The ethanol is then removed by pipetting.
And our DNA has finally been isolated!
Dissolve it in 30µl of T10E1 buffer where individual molecules of DNA separate out and fold properly.
Now we can check the length of our DNA by electrophoresis.



We observe some RNA impurities too. If you really want to prevent this, you could use RNAase during the isolation process- it is an enzyme that degrades RNA.