gel electrophoresis resolution

[Mackenzie Cowell]

Here is a picture of NEB 100bp "quick-load" ladder [1] run in a 1.5% agarose gel run in TBE with GelRed DNA Stain.

The gel was run at 160 V and 100 mA.  It is transilluminated via the setup I previously documented (fotodyne UVC).

Why are the bands of the ladder so wispy?  How can I strengthen them?

Cheers,

Mac

[1] http://www.neb.com/nebecomm/products/productN0467.asp

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[Cory Tobin]

I've never actually seen a gel quite like this one. The tailing of
the bands is usually due to loading too much DNA in the well. But the
bands don't look that bright. Maybe there actually is a lot of DNA
there but since the background light isn't being filtered out it just
doesn't appear very bright?

Some general gel troubleshooting:

- Make sure the the loading buffer is the same as the running buffer.
For example, if you used 1X TAE in the gel and you are using a 5X
loading buffer, the loading buffer should be made with 5X TAE so when
it's diluted in your sample the pH and salt concentration is the same
as 1X TAE.

- Load less DNA

- Replace the running buffer, assuming you have been reusing it
multiple times. Not as critical if you're using TBE.

- Use a lower voltage.

- Fill the chamber lower. 5mm over the top of the gel is ideal.
Excessive buffer leads to smearing.

For more gel debugging and optimization check out a pdf called "The
Sourcebook" by the company Cambrex. http://tinyurl.com/3pqbts8

-Cory

[Mackenzie Cowell]

Cory,

Thanks for the link to the Gel Sourcebook.  Lots of lore.

1) I'm not sure what loading buffer is provided in NEB's 100 bp ladder.  I've just been assuming NEB included some kind of densifying agent, like glycerol.  I have not been adding anything.  All I have been doing is taking 10uL of the ladder for each run.  According to NEB's website, the ladder may be supplied in TAE buffer (http://www.neb.com/nebecomm/products/productN0467.asp) ?

 Not sure.

Also, I have not been adding glycerol to my PCR product prior to loading for electrophoresis.  I have not been adding glycerol because the mastermix I am using obviously already contains loading dye and I wasn't sure if mixing it all with glycerol was necessary.

Interestingly, in the earlier picture of the gel I sent, it looks to me like there is some genomic template in wells 1 and 3 (2 has the ladder), and perhaps the primers rand out to the far right side of the gel in lanes 1 and 3.  The farthest-right band of the ladder is only like 100 bp, so the bands to the right of it are the right size for 20-30 bp primers.

Is that possible?

Mac

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[Mackenzie Cowell]

I've taken a picture of the gel (which I ran about an hour ago and have since kept in the buffer in the gel box) with a UV-blocking acrylic sheet between the gel and the camera.  This should address Cory's idea that background light may have been responsible for the wispiness.  I don't think that's the case, based on the new photo.

Mac

[Cory Tobin]

If you're using the pre-mixed ladder then you are probably loading the
right amount of DNA. When you buy the pure ladder, sometimes people
forget they have to dilute that down at least 10X before using it.
Hence the thought that there may be to much DNA in the well. But
that's probably not the case.

The easiest thing to test would first be the voltage. Drop it down to
90V and see if that works. If it still looks smeared, pour out some
of the buffer so it just barely covers the gel. If that doesn't work,
buy the pure ladder (not the quick load) and mix it with a TBE based
loading buffer.

My guess is one of the first two will fix it.

As for the contents of the non-ladder lanes. Looks like you have two
failed PCR reactions. Primers down at the bottom and genomic?
template at the top. Although, I will say that I've never actually
seen the template show up that bright when loading PCRs in the gel.
My guess is you're adding too much template which can inhibit the
reaction (especially if it was a phenol-chloroform prep). I never put
more than 300ng of genomic prep in the PCR.

-Cory

[sgt york]

1) Too much DNA. It doesn't look very bright because the gel is pretty
heavily stained as well; background is very high.

2) 160V in a 1.5% TBE gel is a bit high, the gel probably overheated a
bit. I'd run at 120V.

3) How thick is your gel? If it's thick, you get a cupping effect on
your bands; the sample forms a bowl-like shape with the open part
facing the well. This happens in every gel, but making the gel thicker
makes it more pronounced.

4) Did the gel ever dry out? It may just be perspective, but the top
and bottom look a little bowed in the picture, like a gel that sat too
long in the open air.

5) You mentioned you didn't add glycerol, but had a pre-added dye in
the PCR. Is this a loading dye? The glycerol in loading dye helps
settle your sample in the well, keeping it tight. If the dye isn't a
loading dye, you need something to load your sample in the well.

First round of things I'd do is load less DNA, pour a thinner gel, and
run the gel slower.

In my experience, loading buffer of the sample & gel being different
doesn't present too much of a problem.

[Emmette Hutchison]

Is this a fresh gel made up in the same TBE running buffer?

Try running at a lower voltage, say around 135V. This might fix the smearing on the ladder, but won't do anything for the DNA stuck in the wells. That's either too much DNA or something funky with it (had a bad batch of klenow cause something similar once.)

Emmette

On Apr 5, 2011 2:40 AM, "Mackenzie Cowell" <m...@diybio.org> wrote:
> Here is a picture of NEB 100bp "quick-load" ladder [1] run in a 1.5% agarose
> gel run in TBE with GelRed DNA Stain.
>
> The gel was run at 160 V and 100 mA. It is transilluminated via the setup I
> previously documented (fotodyne UVC).
>
> Why are the bands of the ladder so wispy? How can I strengthen them?
>
> Cheers,
> Mac
>
> [1] http://www.neb.com/nebecomm/products/productN0467.asp
>

[Simon Quellen Field]

Acrylic is not all that good a UV blocker.

You can tell because the background on your photo is still very blue.

In fact, it looks like the same photo.

Screw a UV Haze filter (they are so cheap people use them just to

protect the camera lens) onto the front of the lens. They stack, so you

can borrow some from friends and screw several on for the test before

you buy six for the project.

You might also look into the specs for UV blocking filters on the web,

as one inexpensive one with good specs might be cheaper than 6 with

poor blocking ability.

Also, get rid of the stuff in the background that reflects UV (the bright

places in your photo).  Zooming in on just the part of the gel you want

will also raise the signal to noise ratio by blocking out more back-

scattered UV.

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[Simon Quellen Field]

Another way to get better signal to noise in the photo is to use

a blue-blocking orange filter.  You want the blue to look black in

the photo.

The Tiffen Haze 2 claims to block all light shorter than 400 nm:

"http://www.bhphotovideo.com/c/product/60729-REG/Tiffen_52HZE2A_52mm_Haze_2A_Filter.html"

or

"http://www.google.com/products/catalog?num=100&hl=en&safe=off&q=Tiffen+Haze+2+price&um=1&ie=UTF-8&cid=2678544646968185094&sa=X&ei=-TibTYfwMInQsAOC4b2LBA&ved=0CD4Q8wIwAw#"

(get the right size for your camera, don't just buy the one I linked to).

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[General Oya]

What about the molarity of the agarose? I know Towson usually runs 1.5, however at CCBC we usually ran .8 M?  These simple changes would also affect resolution as well, no?

Ryan

[Cory Tobin]

Hey Mac, I just saw your thread "identifying reference SNP locations
in sequencing results" and it looks like you got your electrophoresis
working. What did you change to fix the problem?

[I'm posting here to avoid hijacking the snp thread]

-cory