On Fri, Oct 10, 2014 at 6:12 PM, Josiah Zayner <josiah.zayner@gmail.com> wrote:
Yeah, thanks Nathan. Was trying to do something a little more accessible than polyacrylamide. I mean that should totally work though. I was going to order a sequencing kit but instead decided that I might want something a little different and be able to tune my ddNTPs and such so I ordered the chemicals. This is because I think one will probably need more DNA and so instead of doing a cycle sequencing reaction of 20 cycles maybe 200 cycles(it is linear instead of exponential)?
Hmm, I forgot to mention I'd ordered the terminator nucleotides separately, so for a given sample I'd use 4 lanes like radiosequencing gels, so I'd have a way to discriminate bases.
One of the things that sort of got me stuck was lack of funds when I had the time and initiative... but also the chemicals I ordered said something like 'ready to use' but didn't in fact include the polymerization accelerator. Then with being tight on money I wavered from thinking I'd wait to order more items to save on shipping, or something like that, and never ordered anything.
We will see. I think this requires experimentation. I think too many people think, "Oh I can just go sequence this for $3." so no one has really looked much into this avenue or rather published on it. If you have any tips or ideas send them my way.
If polyacrylamide can do single base separation (we know that is true) and that paper I sent shows dinucleotide differences on agarose, then it does seem possible to do some other stuff to push the agarose just that little bit farther. Maybe use purer agarose, or do some in-house cleanup on it (Cathal has posted some good ideas on this in the past) (probably won't beat whatever the super-expensive stuff does), do a really great job at cleaning up your DNA before mixing with the sequencing mix (phenol chloroform or a few rounds of silica adsorption follow by ethanol desalt?), I've also been wondering about pulsed-field electrophoresis as applied to this problem.
I'd think any sanger sequencing would be a minimum of $10-13 for no or minimal prep on their end, not including shipping (I don't think academic labs that are open to public/industry are even setup to mail stuff, so you'd need to drive/bus/taxi over to and from, or get a friend to do it). $3 sounds like pricing that a hospital/govt-lab would pay internally or something, but it'd have to be a huge lab for such prices. Does NASA do it on-campus or outsource or send it across the bay to LBNL/LLNL/JGI?
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