Re: [DIYbio] Borax as a superior alternative to Tris

Proteinaceous buffers would be interesting. After all, BSA is
effectively just a protein buffer, so why not seek and use others?

On agarose biosynthesis, I've looked into it a few times and I can't
find any information on the enzymes responsible. I'm not sure it's been
nailed down, yet. Which is a terrible pity because synthesising agarose
would be awesome!
In the meantime, purifying agarose from agar at home is possible, but
it's not economical without distillation to recover the reagents.

Essentially you just dissolve the agar in propylene glycol at ~105C
(take a long time, and a lot of stirring, and an even/slow heat source
to prevent burning), and then allow to cool to room temperature to
precipitate agarose.

Optionally, then refridgerate/freeze to precipitate more. Then thin out
with an alcohol or acetone (I suggest alcohol to reduce risk of
explosions upon distillation, if that's your route), filter as finely as
possible (coffee filters probably best for this, use several as it's
very viscuous and takes ages). To clean away the propylene glycol,
suspend the filtrate in more alcohol and filter again.

Yields are typically less than 50% of input, and you have to use a lot
of glycol/alcohol to produce it using this method. So, unless you can
safely distill out the two reagents (propylene glycol's boiling point is
over 200C!), it's not worth it.

Another way is to use brewing-grade pectinase enzyme to digest the
agaropectin and allow it to dissolve (takes days): I suspect doing so
before attempting the above would drastically improve yields and reduce
waste. I haven't yet worked up the nerve to devote a whole day to
re-trying the purification. It's messy and very boring.

On 25/06/12 07:47, Meow-Ludo wrote:
> I saw this article<http://www.scq.ubc.ca/the-macgyver-project-genomic-dna-extraction-and-gel-electrophoresis-experiments-using-everyday-materials/> but
> it seems like it could be a bit too budget to get decent results. I also
> found a fair few articles on mixing up TAE yourself, and it seems like it
> may be cheaper.
>
> I wonder if you could get a bacteria to produce enzymes that would make you
> your buffers from something cheaper?
>
> It might also be a good way to produce cheaper agarose too.
>
> On Monday, June 25, 2012 5:53:33 AM UTC+10, Cathal wrote:
>>
>> It would be great to use Borax for other buffers, but I gather the borate
>> ion is inhibitory to enzymes. Tris is a pain to get hold of, so a nice
>> replacement would be really, really cool.
>>
>> On 24 June 2012 14:54, Robert O'Callahan <ropoctl@rice.edu> wrote:
>>
>>> I've used SB buffer to run gels. It's nice to be able to use the maximum
>>> voltage, but they don't run as well. When loading high concentrations of
>>> DNA the migration distance can do weird things, but it works well for
>>> preparatory gels when you need good separation quicker.
>>>
>>> -Rob
>>>
>>>
>>> On Sun, Jun 24, 2012 at 3:54 AM, Cory Tobin <cory.tobin@gmail.com> wrote:
>>>
>>>>> I read a paper a while ago showing a comparison of
>>>>> different buffers, and feel like the borax ones weren't
>>>>> as good for certain fragment sizes or something...
>>>>> what I mean is, I feel like there was some tradeoff
>>>>> between TAE and borax buffers
>>>>
>>>> SB doesn't do well with high molecular weight fragments, like >8k or
>>>> so. TAE works better in that case.
>>>>
>>>>
>>>>
>>>>> But this could possibly made even better by reducing the
>>>>> Na in the solution.
>>>>
>>>> True. Lithium is better but it's expensive.
>>>>
>>>>
>>>>
>>>>> Also, the above papers said that you don't really need
>>>>> EDTA either. The use of EDTA is kind of a left-over
>>>>> from RNA gels that persists in DNA gels for some reason.
>>>>
>>>> This is typical of biology at universities. Someone finds a protocol
>>>> that works and keeps using it without determining what parameters are
>>>> actually important. There is no incentive to try running gels with
>>>> missing components because discovering that EDTA is not needed will
>>>> not get your NIH grant renewed or help you get tenure. From any
>>>> individual's perspective it's better to just continue using protocols
>>>> that work and focus on their research, especially considering that gel
>>>> buffers are a very minor portion of any research budget. Although,
>>>> this might not be the case for diybio.
>>>>
>>>>
>>>>
>>>>> Does anyone here work with Borax? It is readily
>>>>> available from the supermarket and I would be
>>>>> keen to hear of anyone else's experiences
>>>>
>>>> Yeah, it works well. I've used both methods: (1) Borax only and (2)
>>>> NaOH + Boric acid. Both work fine. One thing to note is that you
>>>> can't make a 50X stock of SB like you can with TAE. The max I go is
>>>> 20X and even then I still get some precipitation. It's a bit
>>>> inconvenient but not enough to make me stop using it. Also, if you
>>>> plan on running your gel at 300V for more than 10 minutes, make sure
>>>> you have a box with large reservoirs or you'll melt the gel.
>>>>
>>>>
>>>> -cory
>>>>
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>>>>
>>>>
>>>
>>>
>>> --
>>> Rob O'Callahan
>>> M.S. Civil & Environmental Engineering 2012
>>> George R. Brown School of Engineering | Rice University
>>> E: rpo1@rice.edu<mailto:rpo1@rice.edu>| P: 847.641.1285
>>>
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>>
>>
>>
>> --
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>>
>>
>

--
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