Phenol extraction - (Jul/15/2010 )

Hi everyone !

I'm new to phenol/Chlorophorm DNA extraction, and I tried today with my Boss and we get an interesting, but not wanted, result...
A milk-like solution, without aquaeous/phenolic difference after centrifugation...

What I did :
Ressuspend cells in 4ml of (100mM Tris pH=8, 50mM EDTA pH=8, 50mM NaCl)
Add SDS to final concentration 1%
Add Sarcosyl to final concentration 0,8%

Mix gently by inverting

Adding 4ml of pH8 saturated phenol
Mix several time by inverting


And I get this milk-like solution...
Phenol was yellow.


Any idea ?
Thanks for your help !

You did do a centrifustep after the "mix several time by inverting step" , right?

Yes, 5min at 4kRPM (don't know the g value, need to calculate it)

5 minutes might be too short.

and the speed has to be ok.

Maybe there is something wrong with the mixture at the start, you are sure the chloroform mixture is ok? (phenol/chloroform/isoamyl alcohol 24/1 ratio?)

It was not the phenol/chlorophorm step yet, just saturated phenol.

We were planning to do phenole extraction twince, then phenol chlorophorm twice and to finish, chlorophorm only twice before washind with ethanol.

I note for next time to extand centrifugation time. How much do you suggest ?

I am not really familiar with that protocol. But dont they use TE buffer when working with phenol extraction?

Don't know

I found in an article they are using TE buffer with 100mM NaCl added. I think I will try with this solution.

If anyone have a good idea, I will be pleased to hear it !

Have au good day

Isn't yellow phenol, oxidized phenol?
Do you have a newer bottle?

Maddie on Jul 15 2010, 08:51 AM said:

yup, yellow and as it gets even more oxidized the phenol turns red.

However if 0.1% w/v 8-hydroxyquinoline, a reducing agent used to protect phenol from oxidative damage was added, the phenol solution will turn bight yellow.

From Sluvah protocol I believe only a phenol solution is being used. No chloroform was added, so you can't obtained the aqueous-organic layer.

the composition of phenol/chloroform solution is

25:24:1 = phenol solution:chloroform:isoamyl alcohol

The phenol solution is composed of
10mM TE, 1mM EDTA solution saturated with phenol.

Add 0.1% w/v 8-hydroxyquinoline to your phenol solution, else it will get oxidised very quickly.


For DNA extraction the pH of the phenol solution is pH 8

For RNA extraction the pH of the phenol solution is pH 4.5

perneseblue on Jul 16 2010, 03:25 AM said:

Thats where I was stuck, I couldnt understand it how he wanted to obtain that layer when using only phenol.