Re: [DIYbio] Re: A hypothetical protocol for DIYBIO DNA synthesis

On Sat, Jul 16, 2016 at 10:21 AM, CodeWarrior <code.w4rri0r@gmail.com> wrote:
> lodging it in a pore wouldn't work, it needs to move about to interact with
> the fairly short seed oligos you'd start with.

Right, I said nothing about lodging it.

>
> In an effort to improve the method by finding a way not dependent on special
> reagents like cleanamp dNTPs I've been looking at synthesis by sequencing
> type methods. Consequently I'd like to propose yet another protocol.
>
> 1. a oligo sequence we will call A is introduced to TdT and ordinary dNTPs
> 2. the resulting extended oligos are separated by length

My idea is essentially looping over those two steps, except you do it
recursively (feed output back into input), and stop at some N-number
of length and reserve it for a pool to do sterically-constrained
gibson synthesis (in a reaction chamber less than the polymer
persistence length, so to prevent secondary and tertiary structure
from screwing with ligation). If you command 10-additions with
sufficiently dilute nucleotide solution (dilute enough to
statistically say there is 1 nucleotide per volume introduced to the
reaction chamber), then just discard any molecules that don't meet
your target length (indicating an addition failed or there were more
nucleotides introduced than expected, during a reaction period). You
don't need sequencing either, since you control the input nucleotide.

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