Does Isopropanol freeze? - DNA precipitation (Dec/03/2008 )
Hi there,
I am trying to get the DNA out of my cells, but it seems like that I am running into new issues on a daily basis. Anyhow, I lysed my cells (with CTAB), phenol/chloroform extracted (plus another chloroform extraction) and than precipitate, which is the part that currently doesn't really work. My solution turns milky as soon as I added 100% ethanol. So I decided to try isopropanol instead (and used new salt solutions). The good part was that the solution didn't turn milky after adding isopropanol (plus salt), BUT the sample froze at -80C. I was in a hurry, so I put my samples at -80C rather than -20C. Does isopropanol freeze at -80C? Obviously I am not an expert in DNA precipitation, but I always had the impression that ethanol or isopropanol would stay liquid under those conditions. Or does it not?
Cheers!
I am trying to get the DNA out of my cells, but it seems like that I am running into new issues on a daily basis. Anyhow, I lysed my cells (with CTAB), phenol/chloroform extracted (plus another chloroform extraction) and than precipitate, which is the part that currently doesn't really work. My solution turns milky as soon as I added 100% ethanol. So I decided to try isopropanol instead (and used new salt solutions). The good part was that the solution didn't turn milky after adding isopropanol (plus salt), BUT the sample froze at -80C. I was in a hurry, so I put my samples at -80C rather than -20C. Does isopropanol freeze at -80C? Obviously I am not an expert in DNA precipitation, but I always had the impression that ethanol or isopropanol would stay liquid under those conditions. Or does it not?
Cheers!
The melting point of pure isopropanol is −89 °C, but yours is not diluted...
I added 1 volume of isopropanol to the sample, which would dilute it.... Would it freeze, though (also with the salt in it)? This is the first time I am using -80C.....did you experience the same thing?
I added 1 volume of isopropanol to the sample, which would dilute it.... Would it freeze, though (also with the salt in it)? This is the first time I am using -80C.....did you experience the same thing?
I used only -20°C, it's more than sufficient. But with this melting point it should freeze if diluted.
You could use absolute etoh or isopropanol to precipitate DNA, when added you should see 2 phase a white (milky) one just when you add the alcohol and the clear that is the sample. Just mix both by invertion and you should see the DNA (strings or a white pellet if have a lot of Nuc ac or a change in the viscousity(??) of the mix for a lower quantity of dna(that you will learn to see the difference after a few years of practice)). I recommend that you add at least 2 vol.(up to 2.5vol) of the alcohol. Incubate O/N at -20C. If in a hurry can use it a -80C, but the residues of water in the final supernatant (if any) will make the alcohol to freeze, just leave it in the bench until defrost an then centrifuge.