Ghost bands on plasmid preps (pH determination of phenol)

TFitzwater at Gilead.com TFitzwater at Gilead.com
Fri Apr 6 10:38:18 EST 2001


Re: Ghost bands on plasmid preps
>Nick Theodorakis (nicholas_theodorakis at urmc.rochester.edu)
>Thu 05 Apr 2001 - 15:02:51 BST


>In <20010405094023.31456.qmail at ww02.jatek.com>, Daniel MacArthur wrote:
>
>>Alejandro:
>
>>> Did you perform Phenol-chloroform extraction on your minipreps?
>>> Some years ago I have had the same problem and I solved the
>>> problem shorting the SDS-NaOH step (1-2 min.) and using Phe-chlorof
>>> (I didn't routinely use Phe in my preps).
>
>>No, I used an SDS/NaOH lysis solution, which may well have been the cause
of
>>the problem. If it's not too much trouble, could you give me a little
more
>>detail on the composition of the phenol-chloroform solution? I'd be quite

>>keen to try it out and see what happens.


>I don't believe the phenol/chloroform gets rid of the "form 4" DNA, but I
do use
>it sometimes on minipreps in which I don't use column purification.


>Recipe:
>for crytalline phenol, heat to 60C to melt, saturate with water. Allow the

>phases to separate, remove the water (upper phase), and re-equilibate once
or
>twice with 0.1M Tris pH8; check the pH of the upper phase with pH paper
and make
>sure it's >7. Then remove upper layer and replace with TE. Some people
also
>prefer to add 8-hydroxyquinoline to 0.1% or so to retard oxidation; I have

>noticed thet neutral to basic phenol tends to off a little quicker than
acidic
>phenol. Or just aliquot it and freeze the aliquots.


>Or you can just buy it already equilibrated.


>For phenol:CHCl3, just mix an equal amount of the above phenol and
chloroform.
>Some people prefer to use a 50:1 mix of chloroform and iso-amyl alcohol,
but I
>haven't noticed that it's necessary.


>NB: chloroform dissolves polystyrene plastic; use polypropylene or glass.


>NBB: Phenol is very caustic; use appropriate eye, face, and body
protection when
>handling, especially when equilibrating it.


>I'm also probably obligated to mention that handling open bottles of
chloroform
>outside of a fume hood probably generates air levels of CHCl3 that exceed
OSHA
>standards of safety. But if you wait a few months, the current
adminstration
>might roll back those standards. ;-)


>--
>Nick Theodorakis
>nicholas_theodorakis at urmc.rochester.edu

The indicator chemical in pH paper is destroyed by phenol
resulting in inaccurate pH measurement.

Organics such as phenol and chloroform have much lower dielectric constants
than
water.  The very large liquid junction potential can cause problems such as
pH drift,
long stabilization times and pH electrode damage.   In order to accurately
measure the pH of saturated phenol it is necessary to solubolize the phenol
in an
aqueous medium.

How to pH Tris®-buffered Phenol
For  saturated  phenol  (no  chloroform),  mix 2 mL organic phase with 5 mL
methanol and
13  mL  water and pH entire sample.  The pH will drift down over the course
of about
30  seconds  until  it  stabilizes.   (This  method is from the Ambion 1994
catalog.)

How to pH Tris®-buffered Phenol/Chloroform

For  phenol:chloroform  or  acid phenol:chloroform, mix 2 mL of the organic
phase with
8 mL methanol and 10 mL water.  Mix and measure the pH of the entire sample
with
the  pH  electrode.   The  pH  will  drift down over the course of about 30
seconds until it stabilizes.

pH  measurements of solubolized and unmodified phenol and phenol-chloroform
mixtures
(E. Kleinhenz and S. Cohen 1991 BioTechniques 10(6): 740-741)  (Amresco)
|--------------+------------+------------+------------+------------|
| Unmodified   | Phenol,    | Phenol/CHCl| Phenol,    | Phenol/CHCl|
| organics     | pH 6.6 +/- | 3          | pH 6.6 +/- | 3          |
|              | 0.2        |pH 8.0 +/-  | 0.2        |pH 8.0 +/-  |
|              |            |0.2         |            |0.2         |
|--------------+------------+------------+------------+------------|
| pH paper     |            |            |            |            |
|--------------+------------+------------+------------+------------|
| Hydrion      | 3.5        | 6.0        | 5.0        | 6.0        |
| paper, pH    |            |            |            |            |
| 1-12         |            |            |            |            |
|--------------+------------+------------+------------+------------|
| Hydrion      | 3.0        | 5.0        | 4.0        | 5.0        |
| paper, pH 3-9|            |            |            |            |
|--------------+------------+------------+------------+------------|
| Whatman      | 6.0        | 6.5        | 6.5        | 6.5        |
| paper, pH 5-9|            |            |            |            |
|--------------+------------+------------+------------+------------|
| Riedel-deHaen| 5.5        | 6.5        | 4.5        | 6.5        |
| , pH 0-14    |            |            |            |            |
|--------------+------------+------------+------------+------------|
| EM Science,  | < 6.5      | 6.8        | < 6.5      | 7.2        |
| pH 6.5-10    |            |            |            |            |
|--------------+------------+------------+------------+------------|


|--------------+------------+------------+------------+------------|
| Solubolized  | Phenol,    | Phenol/CHCl| Phenol,    | Phenol/CHCl|
| organics     | pH 6.6 +/- | 3          | pH 6.6 +/- | 3          |
|              | 0.2        |pH 8.0 +/-  | 0.2        |pH 8.0 +/-  |
|              |            |0.2         |            |0.2         |
|--------------+------------+------------+------------+------------|
| Electrode    |            |            |            |            |
|--------------+------------+------------+------------+------------|
| 1            | 6.71       | 8.00       | 6.57       | 8.12       |
|--------------+------------+------------+------------+------------|
| 2            | 6.73       | 7.99       | 6.57       | 8.13       |
|--------------+------------+------------+------------+------------|
| 3            | 6.74       | 7.99       | 6.54       | 8.11       |
|--------------+------------+------------+------------+------------|
| pH paper     |            |            |            |            |
|--------------+------------+------------+------------+------------|
| Whatman      | 6.0        | 6.8        | 6.0        | 7.3        |
| paper, pH 5-9|            |            |            |            |
|--------------+------------+------------+------------+------------|


The electrode used was a Calomel reference electrode (mercury/mercuric acid
reference
cell  type)  obtained  from  Cole-Parmer  (Chicago, IL) model # L-05669-20.
Silver/silver chloride
 type electrodes are not recommended because they do not accurately measure
the pH of
Tris.   Since  the electrode dehydrates during phenol testing, it should be
soaked in Type I
water to rejuvenate the glass bulb.

Tim Fitzwater
Gilead Sciences

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