restriction digest confirmation

[Avery louie]

hello all,

i ran a rather silly gel today.  i wanted to see if a double digest had worked, but the fragments should be about 6 bp and 4kb!  so i obviously was not able to resolve both on my mini gel.

i saw a very large fragment, probably around 4 kb on the gel.  if it were plasmid dna, i would expect it to run as a smaller fragment.  it seems to have worked, but i want to check my reasoning with you all.  obviously, this does not prove both digests worked, but it at least suggests that one did.

if i really needed to make sure it was digested, i would run control single cut reactions against plasmid dna, and the double digest, but i dont have a ton of template dna.

--Avery

[Avery louie]

here is a photo of the gel!

[Avery louie]

top band is a ladder, the largest band is 1.5 kb, and the smallest is 100 bp.  the two lanes beneath it are samples.  the gel was run for about an hour and a half at 60 v and about 50 ma.

[SC]

Hi Avery,

To clarify, the three lanes are ladder, sample1, and sample 2? 

Stacy

 

 

[Avery louie]

yup!

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[SC]

Hmmm... I'm not sure you can tell from this gel.  It's possible that the plasmid DNA is nicked but not cut, so it's running at a higher apparent MW than if it were supercoiled.  

 

Can you extract the band in question from the gel?  Of course, you always lose some when you do that, but you may be able to make another cut to get the bits down to where you can see and size them.

 

 

[Avery louie]

I may try recording the gel "live" as it runs, and looking for the fragment, but I am afraid I won't be able to see it either way.  it is only a few bp long (roughly 6 bp), so what I should really do is run the digest vs the uncut plasmid.

--A

On Tue, Jul 16, 2013 at 12:54 PM, SC <stac...@yahoo.com> wrote:

Hmmm... I'm not sure you can tell from this gel.  It's possible that the plasmid DNA is nicked but not cut, so it's running at a higher apparent MW than if it were supercoiled.  

 

Can you extract the band in question from the gel?  Of course, you always lose some when you do that, but you may be able to make another cut to get the bits down to where you can see and size them.

 

 

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[SC]

The fragment you are looking for is only 6bp?

[Avery louie]

yea, thats the issue.  I am snipping a stop codon+rare codons out of a sequence, and they are basically invisible.

--A

On Tue, Jul 16, 2013 at 2:09 PM, SC <stac...@yahoo.com> wrote:

The fragment you are looking for is only 6bp?

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[SC]

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.  

What is your ultimate goal?  Maybe there will be a different way to proceed.

[Avery louie]

I just wanted to make sure my restriction worked.  running uncut vs cut should reveal what is going on.  I realized after I started it that 6 bp is basically invisible.

--A

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[Nathan McCorkle]

On Tue, Jul 16, 2013 at 11:36 AM, SC <stac...@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

[Avery louie]

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

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[Nathan McCorkle]

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.

> https://groups.google.com/d/msgid/diybio/CAL4KOmggGPUVDj3om%3DmAX5oMDF9F7ZM1YZrdgRZvoTf8ibb%3DsA%40mail.gmail.com.

>
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>
>

--
-Nathan

[Nathan McCorkle]

On Tue, Jul 16, 2013 at 12:37 PM, Nathan McCorkle <nmz...@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,

sorry micromolar, not milli!!!!

[Cathal Garvey (Tablet)]

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..

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[Nathan McCorkle]

On Tue, Jul 16, 2013 at 12:37 PM, Nathan McCorkle <nmz...@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.

Actually, now that I think of it, as long as you use ~1:1 adapter to
template (concentration of all molecules in the digestion, or the
initial template multiplied by 2 assuming there are only two cut sites
associated with your double digestion) and incubate with ligase a
while, you shouldn't see any residual adapters.


OR maybe just assume the double digestion worked, ligate back to
circular, then PCR with a primer that ends with the 6bp site. If it
was deleted, you should get no PCR product.

[Nathan McCorkle]

On Wed, Jul 17, 2013 at 12:29 AM, Cathal Garvey (Tablet)
<cathal...@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?

EtBr looks like it needs a minimum of 2bp
http://en.wikipedia.org/wiki/File:DNA_intercalation2.jpg

and in ssDNA many comments online lead to this general idea:
"According to Sambrook et al, ethidium bromide binds to regions having
an helical character that is mainly caused by intramolecular
interactions. This is not far-fetched since it is known that
single-stranded nucleic acids often form various helical regions, and
this is where ethidium bromide could preferentially bind."

gelGreen and gelRed are much larger molecules, and they look like they
might need mor like 3 or 4 bases (or basepairs). sybr green is pretty
different from them, smaller like EtBr:
http://en.wikipedia.org/wiki/GelGreen
http://en.wikipedia.org/wiki/SYBR_Green_I

"GelRed is structurally closely related to ethidium bromide and
consists of two ethidium subunits that are bridged by a linear spacer"
http://en.wikipedia.org/wiki/GelRed

[Nathan McCorkle]

http://www.nano.uoguelph.ca/manderville/secure/Lecture_3.pdf

--
-Nathan

[Avery louie]

im not sure how big it is, but i am using gelgreen.

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[Ben Gadoua]

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

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[Avery louie]

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

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[Avery louie]

Also,  some of that background is probably reflection.  No translinator.

[Nathan McCorkle]

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)

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[Avery louie]

I think the samples are ok- I may also be at too high of a voltage/temperature.  That ladder is minty fresh from the factory.

--A

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[Cathal Garvey]

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.

[Nathan McCorkle]

On Wed, Aug 7, 2013 at 2:59 PM, Cathal Garvey
<cathal...@cathalgarvey.me> wrote:
> 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.

I hadn't thought of that, I was referring to the edge of the gel being
cooler than the center, so you get smearing in-band unless you use a
capillary where heat transfer is faster thus evening the temperature
difference in the gel greatly. This is one reason why they use 50
micron capillaries for automated Sanger sequencing.