Isolate dNTP

[Antonio Zafra]

I have an important question, how can I isolate dNTP? I think they are basic for using a PCR system.

[Nathan McCorkle]

It will be hard as the 'TP' (triphosphate) is a bit unstable (it
carries the high energy to fuel condensation reactions, i.e.
polymerization), or because most nucleotides in cell lysis solution
could be due to DNA degradation by DNases. You likely would need to
find a protocol to isolate mono or diphosphate nucleotides, then hit
them with a ATP and nucleotide kinase to 'recharge' them.

http://www.jbc.org/content/244/20/5489.full.pdf
http://pubs.acs.org/doi/abs/10.1021/bi00814a011

https://www.neb.com/products/m0201-t4-polynucleotide-kinase
https://www.neb.com/products/m0236-t4-polynucleotide-kinase-3-phosphatase-minus


This is a great project to think about, if you can determine an easy
method for purifying lots of material that can be converted with
kinase to dNTPs then we'd have a cheap DIY source for PCR and maybe
green chemistry DNA synthesis reactions.

On Thu, Feb 13, 2014 at 8:52 AM, Antonio Zafra <azaf...@gmail.com> wrote:
> I have an important question, how can I isolate dNTP? I think they are basic
> for using a PCR system.
>

> --
> -- You received this message because you are subscribed to the Google Groups
> DIYbio group. To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+un...@googlegroups.com. For more options, visit this group at
> https://groups.google.com/d/forum/diybio?hl=en
> Learn more at www.diybio.org
> ---
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To unsubscribe from this group and stop receiving emails from it, send an
> email to diybio+un...@googlegroups.com.
> To post to this group, send email to diy...@googlegroups.com.
> Visit this group at http://groups.google.com/group/diybio.
> To view this discussion on the web visit
> https://groups.google.com/d/msgid/diybio/c99a507b-b916-4715-9c5e-ec6da6d082f1%40googlegroups.com.
> For more options, visit https://groups.google.com/groups/opt_out.



--
-Nathan

[Antonio Zafra]

Thanks Nathan, I think our first interest should be get the principal reagents for PCR. Until we have these reagents we are in "nappies" to manipulate DNA  due to this I mean we need a lot ot things for doing our experiments. I am working in this and in an espectofotometer with arduino. What do you think?

Sorry for my bad english, I am spaniard.

[SC]

Hi Antonio,

 

I applaud your thoughts of generating PCR reagents.  I would like to point out something though:  on a practical level, store-bought dNTPs are not expensive.   It may cost you more to produce them than to buy them. Of course, the process itself may be valuable to you as a learning experience, but if this was intended to save money it may not have that effect.

Stacy

[Cathal Garvey]

I've found the same when it comes to DIY agarose. Plenty of ways of
purifying agar to agarose, but unless you can distil propylene glycol
with your setup (hint: you probably can't!) then the cost of reagents
far outstrips the savings of buying and processing cheap agar.

However, I do think it's important that we have *ways of doing these
things* in the public domain. A little yak-shaving never hurt anyone,
and what if you live somewhere where dNTPs can't be bought for economic,
political reasons? What if you needed some for a critical medical
application?

I can only say that I've heard, anecdotally, that most dNTPs come from
Ireland's fisheries, because they're extracted from salmon sperm. I
don't know whether Salmon sperm is magically rich in dNTPs, or if you
have to process/charge them from dNDPs first.

--
Please help support my crowdfunding campaign, IndieBB: Currently at
23.1% of funding goal, with 27 days left:
http://igg.me/at/yourfirstgmo/x/4252296
T: @onetruecathal, @IndieBBDNA
P: +3538763663185
W: http://indiebiotech.com