Potassium acetate

tfitzwater at gilead.com tfitzwater at gilead.com
Sat Jun 10 21:22:31 EST 2000


>From: Mozart Marins (marins at fcfrp.usp.br)
>Date: Fri 09 Jun 2000 - 13:42:08 BST

>Hi there,
>         Folks, do you know a good site or even a book where I can find
>basic explanations about the function of solutions used during a DNA prep
>such as potassium acetate, ethanol...?

Solution I:  50 mM glucose, 10 mM EDTA (or CDTA) and 25 mM Tris-HCl, pH 8.0
Solution II:  0.2 N NaOH, 1% SDS
Solution III:  3 M potassium acetate, 2 M acetic acid (or 1.8 M formic
acid)

Glucose prevents immediate osmotic lysis of the bacteria and helps prevent
shearing of the DNA.  EDTA disrupts the outer membrane by chelating calcium
(allowing lysozyme to enter, if employed) and magnesium (in order to
inhibit cation-dependent nuclease activity).  CDTA is more soluble than
EDTA in alcohol.  Solution I converts the bacteria to spheroplasts.  Tris
acts as a buffer for Solution II to prevent irreversible denaturation of
ccc (covalently closed circular) DNA above pH 12.6.  Lysozyme degrades the
rigid mucopeptide layer of the cell wall.  RNase A or RNase T1 may be added
to reduce the amount of RNA copurified with the plasmid DNA.

NaOH/SDS lyses the spheroplasts and partially denatures nucleases.  At a pH
of 12.0-12.5, linear plasmid DNA and chromosomal DNA are denatured.  The
ccc DNA is not affected unless the pH is > 12.5.  Proteins are also
denatured at pH 12.0-12.5, eliminating most nucleases.  SDS lyses the cells
and denatures protein.

Potassium acetate is added to pull out cell debris, SDS, SDS-protein
complexes and chromosomal DNA as a white curd-like precipitate.  If added
too quickly, air can be trapped and prevent good sedimentation.  The lower
pH neutralizes the alkali in Solution II and causes the chromosomal DNA to
form an insoluble complex that precipitates in high salt.  The positively
charged potassium ions are used in ethanol precipitation to shield the
negative charge on the phosphate backbone of the DNA.  As a result,
non-ionic, hydrophobic interactions take place which provoke aggregation of
the DNA.  Precipitation at -70 degrees will precipitate out polysaccharides
as well as nucleic acids and should be avoided during plasmid preps.
Potassium acetate is more effective than sodium acetate, but can be
replaced with an equal volume of 7.5 M ammonium acetate.   The acetic acid
is replaced with formic acid (final concentration 1.8 M) when RNases are
used in a subsequent step.

Quantities of the 3 solutions are calibrated to prevent irreversible
denaturation of the ccc plasmid DNA.  Chloroform extraction may be employed
to remove cell wall carbohydrates.  Some protocols employ proteinase K to
eliminate endonuclease I, but proteinase K preps may be contaminated with
nucleases and phosphodiesterases.  Use of an endA- strain of E. coli is
preferable.

Tim Fitzwater
Principal Research Associate
Gilead Sciences


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