Re: [DIYbio] Sanger sequencing DNA/primer amounts -- how and why?

On Tue, Oct 28, 2014 at 10:48 AM, Jeswin <phillyj101@gmail.com> wrote:
> I'm trying to learn more about the sanger sequencing machine we have. I have
> been trying to understand how the "sample submission" guidelines for
> plasmids/PCRs work. Is there a formula for how much template to use and how
> is it calculated?
>
> For example, Genewiz says that for plasmids, you do: (10ng/ul)*(template
> length in kb). For PCR, its (2ng/ul)*(template length in kb). How did they
> come up with those numbers?

Seem like they could be based on the average e.coli gene length and
lab plasmid length. So GFP is ~900bp, and the pGLO plasmid is
~5000bp... 5000/1000 about equals 10 / 2. But they already are taking
into account the template length, so then that logic doesn't fit.
Maybe it is something about the longer pieces being less concentrated,
because the polymerase falls off. There's also the ratio of
terminators to non-terminators to think about, do they mention
changing that for gene vs plasmid? Does the protocol go on to detect
with end-labeling (per fragment there is one dye molecule) or rather
by DNA-associative dyes (per fragment number of dye molecules is
proportional to the length). The latter seems less likely since
co-migrating dye molecules are known to decrease quality of
electrophoresis, and they're probably doing capillary gels so
post-staining is not happening (since the fragments are only watched
for at the end of the capillary, and the capillary is glass).

So it seems to be something about the enrichment, I tried finding PCR
amplicon size distribution plots/data, but came up short, though I
didn't spend too long searching. Hope this will give you some help to
dig up more info!

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