PEG

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Fri May 26 10:45:51 EST 1995


zoommmm at aol.com (ZOOMmmm) asks:

> How does PEG work in the process of purifying DNA?

I'd like to expand some of the points found in the thread
that developed in response to this question.

1)  PEG forms large random coils that include water but exclude
    DNA.  The DNA eventually precipitates from lack of solvation.
    MW of the DNA, MW of the PEG, and salt conc. all matter.
    You can do crude size cuts by sequential centrifugation
    with increasing concentrations of PEG.

2)  Other macromolecules also precipitate, each at their characteristic
    PEG concentration.

3)  Though some PEG gets caught up in the pellet, most of the PEG is
    left behind in the supernatent.  So the "PEG pellet" is really
    not a pellet of PEG.

4)  PEG that carries over generally does not directly interfere with
    subsequent reactions.  In fact, a little PEG usually is benificial
    due to its molecular crowding effect causing concentration of the
    substrates.  However, people often discover that PEG precipitants
    appear badly inhibited for further reaction.  This is because of the
    other non-specific crud that was co precipitated with the DNA.
    PEG itself can be easily removed with chloroform extraction; but, of
    course, getting rid of the crud will depend on exactly what the crud
    is.

5)  If your DNA ends up in this condition, then you may need to 
    fall back to whatever purification procedures you would have used
    instead of PEG in the first place.   

6)  Some specific sources of trouble are:
    a) Carrying over a lot of the supernatent with the pellet, hence
       including a lot of whatever you were trying to remove in the 
       first place. This is one of the most common failings when
       PEG is used as a fast and dirty prep. for sequencing templates.

    b) Using too much PEG or too high a MW PEG, hence bringing down
       stuff you were trying to leave behind.

    c) Using too low a MW PEG, leaving your DNA behind.

    d) Failing to remove all that NaCl you included in the PEG precipitation.
       (The old big white pellet after EtOH prec. problem.)

    e) Expecting it to quantitatively remove a contaminant.  You use this step
       when you want to concentrate the DNA (or phage) and knock down
       smaller stuff (PCR primers, bulk protein, tRNA, etc.) a couple of orders
       of magnitude.  It's not appropriate for contaminants where a 1%
       carry-over would compromise your experiment (ie. enzyme activities).

    f) Expecting it to remove stuff that precipitates as well as your
       DNA (ribosomes, cell debris, chromosomal DNA, shards of agarose
       or polyacrylamide).

7) When used in an appropriate context, this method is relatively convenient
   and forgiving of technical imprecisions of most kinds.

Steve Hardies, Assoc. Prof. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu





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