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

Isn't BSA just used to bind non-specifically during preps, and maybe
cause molecular crowding a bit for some reactions?

On Mon, Jun 25, 2012 at 4:34 AM, Cathal Garvey <cathalgarvey@gmail.com> wrote:
> 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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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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