Re: [DIYbio] Re: restriction digest confirmation

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Indeed; with flat-bed gels in particular, if there isn't enough buffer
atop the gel, the voltage tends to short-circuit through the liquid
buffer and disproportionately heat the top millimeter of the gel,
sometimes melting and shifting it along with any DNA contained within.

At least, so spake the authors of the paper that originally recommended
high-impedence buffers like Sodium Borate.

If so, you can either add more buffer to the gel tank, but my
understanding is that you're then in effect simply reducing the
proportional voltage across the gel itself, or reduce the voltage, or
reduce the DNA you add so that it sits low in the well and doesn't
actually enter the top millimeter of the gel in significant quantities.

Probably easiest, though most time consuming, is just to reduce voltage
and see if that helps.

On Mon, 5 Aug 2013 12:24:06 -0700
Nathan McCorkle <nmz787@gmail.com> wrote:

> Gel % shouldn't cause smearing. Smearing is either wonky heat related
> issues (heat changes electrophoretic mobility) or ion contamination,
> or degradation of the amplicon, or that the amplicon had a wide
> distribution of lengths to begin. (From my experience)
>
> On Mon, Aug 5, 2013 at 9:44 AM, Avery louie <inactive.e@gmail.com>
> wrote:
> > I agree, but thats all I am trying to do. I think the secret is
> > the gel is too high %. I made up a large stock of it before we had
> > a decent scale, sovi am getting a lot of smearing.
> >
> > Also, my phone camera makes our look much worse.
> >
> > --A
> >
> > On Aug 5, 2013 12:37 PM, "Ben Gadoua" <ben.gadoua@gmail.com> wrote:
> >>
> >> To be frank, that gel looks awful. Can we have your detailed
> >> procedure, and materials?
> >>
> >> Looks like there was a lot of background gelgreen in the gel
> >> itself, and your ladder is pretty hard to make out. You probably
> >> have confirmed your double digest but it might be worth it to take
> >> a look at your procedure to see if there's anything you can do to
> >> make gels come out more clearly. If you were doing anything other
> >> than looking for just one segment that gel would be actually
> >> useless.
> >>
> >> Ben Gadoua
> >>
> >>
> >> On Wed, Jul 17, 2013 at 5:21 AM, Avery louie
> >> <inactive.e@gmail.com> wrote:
> >>>
> >>> im not sure how big it is, but i am using gelgreen.
> >>>
> >>> On Jul 17, 2013 3:20 AM, "Cathal Garvey (Tablet)"
> >>> <cathalgarvey@cathalgarvey.me> wrote:
> >>>>
> >>>> How big, basepair wise, is gelred, or EtBr for that matter? At
> >>>> 6bp I'd be wary that normal intercalating dyes might interrupt
> >>>> your DNA duplex?
> >>>>
> >>>> Also, more DNA would improve resolution of your fragment, but
> >>>> that much DNA might also reduce cutting efficiency of your
> >>>> enzymes..
> >>>>
> >>>> Nathan McCorkle <nmz787@gmail.com> wrote:
> >>>>>
> >>>>> So just do the math to bump the fragment up to the minimum
> >>>>> detection concentration by adding more template... or after
> >>>>> digestion, ligate on adapters (primers) to bump up the mass of
> >>>>> ANY/ALL fragments. In the
> >>>>>
> >>>>>
> >>>>>
> >>>>>
> >>>>> latter case, you'd then need to differentiate 6bp.
> >>>>>
> >>>>> (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
> >>>>>
> >>>>>
> >>>>>
> >>>>>
> >>>>> (500*6)*((3*10^-6)*1/1000000)/100*10^9
> >>>>> =0.09 nanograms
> >>>>>
> >>>>> this is assuming your pre-digested plasmid concentration was 3
> >>>>> millimolar, and you added 1uL to the digest. This doesn't take
> >>>>> into account the size of your gel well, which will influence the
> >>>>>
> >>>>>
> >>>>>
> >>>>>
> >>>>> concentration of DNA in the band. But since gelRed/gelGreen
> >>>>> doesn't give dimensions in their datasheet, we've got to assume
> >>>>> that the well is the same size.
> >>>>>
> >>>>> Since
> >>>>> gelRed says "Some users have reported being able to detect less
> >>>>> than 0.1ng DNA." That tells me that you might be right on the
> >>>>> cusp of sensitivity, though if your gel well is 4 times more
> >>>>> voluminous than
> >>>>>
> >>>>>
> >>>>>
> >>>>>
> >>>>> the gelRed people's etc, your concentration would be less than
> >>>>> the minimum.
> >>>>>
> >>>>> On Tue, Jul 16, 2013 at 12:07 PM, Avery louie
> >>>>> <inactive.e@gmail.com> wrote:
> >>>>>
> >>>>>
> >>>>>
> >>>>>
> >>>>>> Differentiated, but have you ever seen 2bp? The problem is not
> >>>>>> resolution,
> >>>>>> it is a mass problem. That the fragment is 6bp, and if you
> >>>>>> start out with 1
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>> ug of dna that is 4kb long, then the weight of the 6bp
> >>>>>> fragment is 6/4000
> >>>>>> ug, which is pretty small (1.5ng) and totally invisible. For
> >>>>>> reverence, i
> >>>>>> used 1 ug of dna for my digest, and used 1/5 of the digest for
> >>>>>> the gel, so
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>> we are down to .3 ng. Waaaayyyy below my ability to detect.
> >>>>>>
> >>>>>> --A
> >>>>>>
> >>>>>>
> >>>>>> On Tue, Jul 16, 2013 at 2:44 PM, Nathan McCorkle
> >>>>>> <nmz787@gmail.com> wrote:
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>
> >>>>>>> On Tue, Jul 16, 2013 at 11:36 AM, SC <stacy734@yahoo.com>
> >>>>>>> wrote:
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>>> Ah. My apologies, you did mention that in your orginal
> >>>>>>>> post, but my brain
> >>>>>>>> saw "6 kb" instead of "6 bp".
> >>>>>>>>
> >>>>>>>>
> >>>>>>>>
> >>>>>>>>
> >>>>>>>>
> >>>>>>>> I don't think you would ever be able to see such a small
> >>>>>>>> fragment on an
> >>>>>>>> agarose gel.
> >>>>>>>
> >>>>>>>
> >>>>>>> I've seen as small as 2bp differentiated on agarose, but you
> >>>>>>> need to be careful about your reagents and use a really high
> >>>>>>> % gel
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>> http://diyhpl.us/~nmz787/pdf/Agarose-Based%20System%20for%20Separation%20of%20Short__97225pf01_10994a.pdf
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>>
> >>>>>>>
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> >>>>
> >>>>
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