Re: [DIYbio] Re: Plant Engineering

I've dissociated chicken heart before using trypsin, but they don't
have a cell wall so their main cell membrane may naturally be tougher.
I've also fused B cells before to make hybridomas and you've got to be
very careful with that, they're very susceptible to rupture. Same with
spheroplasts/protoplasts in e.coli... very fragile. I would imagine
you could just add whatever *-ase to some buffered media or even
growth media and wait, maybe stir gently with a pipet tip every 10
minutes for 30-60 minutes. Vortexing once a minute for 5 minutes would
also be a good 'other end of the gentleness spectrum' test to try.

got this by googling protoplast preparation, looks pretty good, says
to use shaking incubator for an hour or so
http://ivaan.com/protocols/128.html

here are relevant links too
http://en.wikipedia.org/wiki/Protoplast
http://en.wikipedia.org/wiki/Spheroplast

This looks OK too, $71 USD for 50mL
http://www.sigmaaldrich.com/catalog/product/sigma/V2010?lang=en&region=US

Viscozyme® L - A product of Novozyme Corp
General description:
Multi-enzyme complex containing a wide range of carbohydrases,
including arabanase, cellulase, β-glucanase, hemicellulase, and
xylanase


On Thu, Nov 1, 2012 at 2:42 PM, Andreas Sturm <masterstorm123@gmail.com> wrote:
> Ah thanks.
> Looked up cider, the german article in english say that cider is not
> wide-spread in Austria, we mainly have "Most" (which is closely related).
> http://de.wikipedia.org/wiki/Most_%28Getr%C3%A4nk%29 for Most, there seems
> to be no adequate english homology, as there's not even a translation.
>
> Doesn't relly matter, though, I don't drink that kind of stuff, neither
> beer, cider nor wine ;)
>
>
> When applying just the cellulase and then vortex, will this set free at
> least some cells? I'm gonna try that out anyway ;)
>
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> On Thu, Nov 1, 2012 at 10:33 PM, Cathal Garvey <cathalgarvey@gmail.com>
> wrote:
>>
>> Brewer's pectinase should work, but I imagine it's less pure than the
>> lab-grade stuff (if someone has access to both, a comparison would be
>> awesome!)
>>
>> I've been thinking for a while now about bugging someone with more
>> protein-fu than I to help codify a method of separating cellulase from
>> those hippy digestive aid tablets you find in good hippy healthfood
>> stores; they often come with pectinase, cellulase, etc. etc., but also
>> lipase and a bunch of other potentially lethal *ases which render them
>> useless for plant protoplast engineering, most likely. A set of steps
>> you could follow to precipitate out unwanted proteins or otherwise
>> purify the cellulase/pectinase from these tablets/powders would be great
>> for DIY plant engineers!
>>
>> Anyways, as far as viability is concerned trust the packaging. If you
>> need to test if your pectinase is working, you could always add some to
>> cloudy apple juice and see if it clarifies in a few hours or days! :)
>>
>> Also FYI: in English Apple/Pear ferments are usually called "cider".
>>
>> On 01/11/12 14:31, Mega wrote:
>> >
>> >
>> >
>> > I'm gonna try to make protoplasts within the next week. I've got
>> > cellulase
>> > from my university, but there seems to be no pectinase left.
>> >
>> > Can anyone guesstimate if this pectinase will work?
>> >
>> > http://www.ebay.at/itm/Pektinase-Enzym-Maischeverflussiger-50gr-/150737056744?pt=Spezial_Landwirtschaft&hash=item2318a0f3e8
>> >
>> > It's for when you want to brew "beer" from apples and pears (we call
>> > that
>> > "fruit beer" Most in German).
>> >
>> >
>> > And, especially: for how long the pectinase powder will work? Can I keep
>> > it
>> > 1 year without decaying? Because there's a lot of it in this package
>> > (that's fine because of shipment, but i it decays too quickly, it's
>> > wastement)
>> >
>> > thanks ;)
>> >
>>
>> --
>> www.indiebiotech.com
>> twitter.com/onetruecathal
>> joindiaspora.com/u/cathalgarvey
>> PGP Public Key: http://bit.ly/CathalGKey
>>
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--
-Nathan

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