Re: Get a plasmide - buy? share?

Lysozyme from eggs? that sounds great!! Do you have a protocol/
instruction about this?

Do I just have to mix egg (the yellow and the white piece - both of
them?) with ethanol and kind of 'shake' it (using a kitchen
machine)?
Aren't there any other proteins/enzymes that will be soluted by the
ethanol??

On 1 Jan., 11:10, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Actually, one of the reasons alkaline preps work so well for plasmid
> isolation is the fine balance of alkalinity between "denatures DNA" and
> "Denatures *even supercoiled DNA*". Because plasmids are usually found
> in some form of supercoiling within cells, they are tightly wound up and
> resistant to denaturing. The high pH of the lysis/denaturing buffers in
> minipreps force protein, fat *and* chromosomal DNA into an insoluble
> mass which can be easily removed, while leaving plasmids intact.
>
> So, provided you could precisely hit the target pH in one swift step,
> you could do the miniprep with just Sodium Hydroxide as in the youtube
> video. However, that would be pretty prone to error!
>
> Also, you can do without lysozyme for lab strains of E.coli, and other
> gram negatives may burst just because of the high pH. However, I
> wouldn't rely on that to work with gram positives; they have just too
> much peptidoglycan to burst in the needed time frame. By the time the
> sodium/potassium hydroxide chewed them open, it would probably have
> damaged the DNA quite a bit also.
>
> Lysozyme's cheap to buy from brew-shops, and you can prepare it from
> eggs using cheap vodka, do might as well use lysozyme. If you're
> prepping your own, only use free-range eggs; not only is it nicer to the
> hens, but they tend to have far more lysozyme in them.
>
> On 31/12/11 20:29, Nathan McCorkle wrote:
>
>
>
>
>
>
>
>
>
> > 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.
> > On Dec 31, 2011 8:30 AM, "Mega" <masterstorm...@gmail.com> wrote:
>
> >> @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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