Problem with phenol
hroychow at NMSU.EDU
Fri Jun 16 17:32:53 EST 1995
On Fri, 16 Jun 1995, Ramachandran wrote:
> Is there anyone who can tell me about the kind of phenol that should be
> used for isolating genomic DNA from mouse tails. Is there some kind of
> time tested special method to buffer the phenol?
> And I have been trying to bring down the pH of phenol (to be used in DNA
> isolation) with several washes of 0.1 M Tris-Cl, without any success. I
> have done almost 7 washes with 0.1 M Tris-Cl. I am following the
> protocol given in the Maniatis Lab mannual. If someone can suggest a
> solution it will be great!
> Thank you,
> Institut fuer Molekularbiologie II
> der Universitaet Zuerich
> Winterthurerstr. 190
> CH 8057, Zuerich
> e-mail: ramc at molbio2.unizh.ch
The type of phenol that one uses in the lab is very much dictated by
economics. If the lab has ample money, you could simply buy the purest
form of re-distilled compound from one of the major suppliers. If not,
then you could buy the not-so-pure chemical and distill it yourself in the
lab. We did it once a year in my PhD lab. It was not fun.
Are you sure that you are trying to bring down the pH of the Phenol?! One
long equilibration with 1M Tris, pH8 and two relatively shorter ones with
0.1M Tris, pH8 - that's all, and your equilibrated phenol should be
saturated with the buffer. So, how do you suppose that the pH will go any
lower than that of the Tris buffer! Although you did not mention the pH
of the buffer you are using, I am assuming that you are titrating your
buffer to the correct pH (which should most certainly be above 7.5). And
I hope that by Tris.Cl you do not mean (and use) only TrisHCl. There has
been some discussion lately about this on the net.
All that aside, the fact is that the NA's will partition with the phenol
at acidic pH and that's something you do not want. What do you think the
purpose of this equilibration is? .... Phenol, a.k.a. phenyl
hydroxide/hydroxybenzene, is also known as Carbolic acid/phenic
acid/phenylic acid - it is already acidic - and that is why you equilibrate
with Tris, pH8.0.
Hiranya S. Roychowdhury
Plant Genetic Engineering Lab.
Box 3GL, NM State Univ.
Las Cruces, NM 88003
Phone: (505) 646-5785
hroychow at nmsu.edu
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