Re: [DIYbio] Re: restriction digest confirmation

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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