Re: [DIYbio] Re: restriction digest confirmation

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
>>
>> --
>> -- You received this message because you are subscribed to the Google
>> Groups DIYbio group. To post to this group, send email to
>> diybio@googlegroups.com. To unsubscribe from this group, send email to
>> diybio+unsubscribe@googlegroups.com. For more options, visit this group at
>> https://groups.google.com/d/forum/diybio?hl=en
>> Learn more at www.diybio.org
>> ---
>> You received this message because you are subscribed to the Google Groups
>> "DIYbio" group.
>> To unsubscribe from this group and stop receiving emails from it, send an
>> email to diybio+unsubscribe@googlegroups.com.
>> To post to this group, send email to diybio@googlegroups.com.
>> Visit this group at http://groups.google.com/group/diybio.
>> To view this discussion on the web visit
>> https://groups.google.com/d/msgid/diybio/CA%2B82U9LJjyutiRVhVawRJ41skPbs44e2yAk5XZCERA2%2BqpiMCA%40mail.gmail.com.
>>
>> For more options, visit https://groups.google.com/groups/opt_out.
>>
>>
>
> --
> -- You received this message because you are subscribed to the Google Groups
> DIYbio group. To post to this group, send email to diybio@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+unsubscribe@googlegroups.com. For more options, visit this group at
> https://groups.google.com/d/forum/diybio?hl=en
> Learn more at www.diybio.org
> ---
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To unsubscribe from this group and stop receiving emails from it, send an
> email to diybio+unsubscribe@googlegroups.com.
> To post to this group, send email to diybio@googlegroups.com.
> Visit this group at http://groups.google.com/group/diybio.
> To view this discussion on the web visit
> https://groups.google.com/d/msgid/diybio/CAL4KOmggGPUVDj3om%3DmAX5oMDF9F7ZM1YZrdgRZvoTf8ibb%3DsA%40mail.gmail.com.
>
> For more options, visit https://groups.google.com/groups/opt_out.
>
>



--
-Nathan

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diybio@googlegroups.com. To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+unsubscribe@googlegroups.com.
To post to this group, send email to diybio@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/CA%2B82U9%2B1__jwT4kd4EsVjrDqSjstY9eYMCyt1gQE1xJDG6%2BaiQ%40mail.gmail.com.
For more options, visit https://groups.google.com/groups/opt_out.