Re: [DIYbio] 10% Agarose and 1-2 nucleobase resolution

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)?

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.

Thanks.

On Fri, Oct 10, 2014 at 5:35 PM, Nathan McCorkle <nmz...@gmail.com> wrote:

I've read up on this before, and even wrote up a wiki page on how I'd go about accomplishing a mini-sequencing-gel... but the basic idea was that a dye like gelRed had pretty good sensitivity, and this is based on the total number of bases available for intercalation/association.

So you start your sanger sequencing reaction with lots and lots of primer, such that each pool of amplicons is very dense, and since these co-locate during electrophoresis, you'd get a nice bright signal.

Here's the paper I was basing the general electrophoresis protocol off of, but I'd decided to try polyacrylamide since I was looking for nice-looking gel images:

http://www.biotechniques.com/multimedia/archive/00010/97225pf01_10994a.pdf

I ordered a sanger sequencing kit and got the polyacrylamide, but then got busy or something (and I don't have much biohacking community around for these projects to progress without my full commitment).... so I never tested it.

The basic math I did for the required starting primer concentration to get high enough signal density was:

nanograms_DNA_in_band = (formula_weight_of_nucleotide * length_of_DNA_in_band) * (concentration_of_limiting_reagent * microliters_of_reagent_added / num_microliters_in_liter) / num_bands_expected * num_nanograms_in_gram

nanograms_DNA_in_band needs to be larger than the dye's datasheet specs for minimum sensitivity.

On Thu, Oct 9, 2014 at 3:42 PM, Josiah Zayner <josiah...@gmail.com> wrote:

So at Science Hack Day SF I worked with a bunch of people to attempt to perform DIY DNA sequencing without any expensive equipment or toxic chemicals.

We were not able to visualize the sequencing reactions yet(don't know if they worked). However, we were able to achieve ~1-2 nucleobase resolution in a 10% agarose gel.

Here is a blog post about it: http://doitourselfscience.blogspot.com/2014/10/science-hack-day-sf-2014-diy-dna.html

Hopefully in the next few months I plan to continue the work at Biocurious.

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