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:
--
-Nathan
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.zayner@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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-Nathan
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