Re: [DIYbio] Re: restriction digest confirmation

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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>>>>>
>>>>> --
>>>>> -Nathan
>>>>
>>>>
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--
-Nathan

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