Responses

Vance Baird vance_baird at QUICKMAIL.CLEMSON.EDU
Thu Sep 30 19:44:56 EST 1993


                       Subject:                               Time:7:36 PM
  OFFICE MEMO          Responses                              Date:9/30/93
This is a summary of responses to my ? about the importance of the pH of the
acetate neutralization solution in the "alkali pDNA prep".  Since no one had a
definet reason for choosing a specific pH, I assume it is important only to the
extent that "neutralization" is accomplished completely/rapidly.  Thanks.
Anyone disagree with, or want to add anything to, the following?

Alkali denaturation:  Basically you are trying to isolate and purify pDNA
(~covalently-closed-circular dsDNA) away from bacterial chromosomal DNA, RNA,
proteins (nucleases), polysaccharides, lipids, etc.

To do so you lyse the cells with SDS (cell wall may be weakened by
pre-treatment w/lysozyme), and simultaneously denature the chromosomal DNA with
alkali (or heat) to its single stranded form.

Then you rapidly neutralize the solution, in the presence of high [salt].  This
allows (any denatured plasmid to reanneal, "snap-back") the linear chromosomal
DNA to form an insoluble protein-DNA-SDS precipitate, and then remove it by
centrifugation.  This may be followed by further deproteinization (e.g. phenol)
and EtOH or iso-propanol ppt.; but pDNA may be clean enough for most puposes
w/o phenol extraction.  However, good washing (2nd ppt.) of the pDNA pellet may
be necessary.


Glucose in the resuspension solution serves as a pH buffer.

EDTA in the resuspension buffer helps permeablize the outer membrane of the
bacterium.  More permiable to SDS, ,which lysis membranes and denatures
proteins, (and lysozyme, which degrades bacterial wall).

Proper pH (12.0 to 12.6) for denaturation (sol. #2) is acheived by maintaining
an "appropriate" ratio of cell suspension to NaOH.  A pH above 13 will
irreversable denature CCCpDNA; while exceeding the 5-10 minute denaturing time
risks "nicking" the pDNA (CCCpDNA usually runs just ahead of the
nicked-circular pDNA, fastest band, on an agarose gel using TBA running buffer)

Chromosomal DNA is selectively denatured, and then precipitates as an insoluble
network (ss and "random" dsDNA) following rapid neutralization with acidic K-
(or Na-) acetate.  The larger (i.e., higher mol. wt.) the chomosomal DNA is,
the better it "precipitates"; so avoid shearing it in the early stages when
adding and mixing solutions.

Simultaneously the high [acetate] (K-form is more effective) induces the
precipitation of SDS (anionic detergent) denatured proteins and high molec.
weight RNA.





More information about the Arab-gen mailing list