Re: [DIYbio] Miniprep without EDTA

One more note about the KCl: the KCl is only used to precipitate the
SDS which subsequently traps the cell debris including the genomic
DNA, allowing it to be separated by centrifuging (sodium dodecyl
sulfate is soluble in water whereas potassium dodecyl sulfate is
insoluble). The NaCl is used in the next step along with the vinegar
to bind the DNA. DNA will bind to silica (diamonaceous earth) when
NaCl > 2M and 4 < pH < 6*. If you could find a way to lyse cells and
remove the debris without SDS then you could eliminate the KCl.

Regarding the vinegar: in this protocol the only purpose of the
vinegar is to get the pH down to 4.5. In commercial kits, the acetic
acid forms a buffer with the potassium acetate (weak acid + conjugate
base), but I've found the pH range in which DNA binds silica is quite
large so getting a precise pH is not very critical. I tried
substituting HCl since you can get pure HCl (37% in water) from
swimming pool supply stores under the name "muriatic acid" but since
HCl is a strong acid, getting the pH within the acceptable range
without a buffer was complicated. Other weak acids might be
acceptable substitutes for vinegar.

Finally, an anecdote regarding the NaCl:

All of the commercial kits I am familiar with use guanidine
hydrochloride to make the DNA bind to silica. Most of the literature
on DNA purification revolves around the use of chaotropic salts like
guanidine hcl, but those chemicals are expensive, often toxic and are
only available through chemical distributors.

One time the lab that I work in had received a shipment of defective
Qiagen kits, so instead of waiting for Qiagen to fix the problem I
mixed my own solutions using guanidine hcl. I noticed the solutions
with guanidine had properties similar to water saturated with NaCl,
like low surface tension and low adhesion to plastic (while pipetting
the solution tends to drip out of the tip instead of staying in the
tip during transfer). So I Googled/Pubmeded for any reports of NaCl
being substituted for guanidine and found only a single paper (Lacksmi
et al, 1999, Analytical Biochemistry) that used NaCl for minipreps.
Apparently the use of inexpensive NaCl has been overlooked by the
miniprep kit manufacturers. Sucks for the thousands of labs who have
been overpaying for guanidine for the last decade, but good for
DIYers.


*The pH 4-6 range is somewhat nebulous. I haven't explored the pH
dependence thoroughly so the boundaries might actually be somewhat
larger or smaller than that.


-cory

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