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" <masterstorm123@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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