Re: [DIYbio] Miniprep without EDTA

It's also a powerful denaturant, so having guanidine in the lysis/neutralisation buffer is handy for that reason, too. Helps kill tough proteins like nucleases, and reduces waiting time. Another common chaotropic salt is urea, which should have very similar properties to guanidine hcl.

However, for convenience nothing beats salt! That's an awesome hack/discovery I wasn't aware of, thanks!

Cory Tobin <cory.tobin@gmail.com> wrote:

>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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