An alkaline miniprep is basically an acid/base (A/B) extraction where the point is to "defat" the cell solution using detergent to act as the nonpolar component... it diverges from a normal A/B extraction because you don't separate nonpolar from polar before neutralizing... thanks to the size of chromosomes/nucleosomes they get caught up with the nonpolar stuff and pellet out.
Lysozyme isn't needed, but helps. Sugar in buffer is probably just for osmolarity so cells don't burst before being alkalinized, because the alkalinity deactivates nucleases. Triton-X is a line of fancy and well documented detergents, so protocols have been refined to make sure its all cleaned from the plasmid solution... other detergents will work, but the protocol may be lossy without lots of experimental optimization. EDTA is also there to stop nuclease activity but again not absolutely required... unless you plan on storing the plasmid for a while or at room temperature.
Sent from my mobile Android device, please excuse any typographical errors.
@Ethan,
Ok, so its not that easy.... It's a pitty!
I won't need the chromosomal DNA & nuclease digesting my plasmids is
also terrible...
On 27 Dez., 20:09, Mega <masterstorm...@gmail.com> wrote:
> Will I need lysozyme or will the STET-buffer wwill be sufficient for a
> boiling lysis??
>
> (I'll try to get the chemicals now... Just STET, isopropanol/ethanol
> and water?)
>
> On 27 Dez., 18:13, Cathal Garvey <cathalgar...@gmail.com> wrote:
>
> > Correction: 300mM. I blame Christmas fatigue for my unreliable literacy.
>
> > Cathal Garvey <cathalgar...@gmail.com> wrote:
> > >By the way, a reliable source tells me that 100mM of sodium chloride is
> > >just as good as sodium acetate. As table salt is cheaper than
> > >handwarmer filling, this is helpful to know.
>
> > >You may want to search for 'pure' salt though, without iodine,
> > >preservatives or anticaking agents, as these may affect the
> > >outcome.iodine in particular would strike me as a dodgy thing to add to
> > >a DNA prep, it can be pretty reactive right?
>
> > >Jeswin <phillyj...@gmail.com> wrote:
>
> > >>On Tue, Dec 27, 2011 at 9:55 AM, Mega <masterstorm...@gmail.com>
> > >wrote:
> > >>> In our university's lab there are big ammounts of 'pure' ethanol
> > >>> (97%). I think isopropanol is more difficult to obtain for me.
>
> > >>Isopropanol is also known as "rubbing alcohol". You can find high
> > >>percentage, above 70% and maybe 80% or 90%, at your local drugstore. I
> > >>would think it it is much harder to procure ethanol.
>
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