Separating PCR products on agarose gel with similar bp length

[umwo...@cc.umanitoba.ca]

Hi,

I am trying to separate 745bp, 646bp, and 655bp fragments on an
agarose gel. I've separated the 745bp fragment but the other two
fragments are not separating on a 2.5% agarose gel, running at 55V for
3.5 hours. I also want a crisp band because I am planning to cut these
fragments out and extract the DNA for sequencing. Any suggestions?

[Peter Ellis]

Would it be simpler to clone the mixture and then just sequence half a
dozen clones until you get the right one? Or is the band you want much
weaker than the others?

Peter

[Taliaferro, Dwayne (NIH/NIMH) [F]]

Run a larger gel (more distance between the + and - ends) overnight with 3% agarose. You could also try a polyacrylamide gel as well, but I don't have experience running one for that purpose. If its appropriate I'm sure someone will suggest the proper % and setup apparatus.

On 12/4/09 4:17 PM, "umwo...@cc.umanitoba.ca" <umwo...@cc.umanitoba.ca> wrote:

Hi,

I am trying to separate 745bp, 646bp, and 655bp fragments on an
agarose gel. I've separated the 745bp fragment but the other two
fragments are not separating on a 2.5% agarose gel, running at 55V for
3.5 hours. I also want a crisp band because I am planning to cut these
fragments out and extract the DNA for sequencing. Any suggestions?

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Methods mailing list
Met...@net.bio.net
http://www.bio.net/biomail/listinfo/methods

[Cathal Garvey]

If you know the sequence of the bands, check if you can cut one of them
using a restriction enzyme so that it forms two smaller bands instead.

[umwo...@cc.umanitoba.ca]

Hi Peter,

Thanks for the suggestion but due to recent events the lab I work at
does not have the capabilty to do cloning. I also want to see if there
are any other similar sized fragments (possible splice variants) in
the amplified region of the gene.

[Rosario Díaz-González]

That's a good suggestion, and I can give a tip for settings: try 2V/cm of
agarose gel length for overnight running. It should work nicely.

On Fri, 4 Dec 2009 17:26:31 -0500, Taliaferro, Dwayne (NIH/NIMH) [F] wrote

> Run a larger gel (more distance between the + and - ends) overnight
> with 3% agarose. You could also try a polyacrylamide gel as well,
> but I don't have experience running one for that purpose. If its
> appropriate I'm sure someone will suggest the proper % and setup apparatus.
>
> On 12/4/09 4:17 PM, "umwo...@cc.umanitoba.ca"

> <umwo...@cc.umanitoba.ca> wrote:
>
> Hi,
>
> I am trying to separate 745bp, 646bp, and 655bp fragments on an
> agarose gel. I've separated the 745bp fragment but the other two
> fragments are not separating on a 2.5% agarose gel, running at 55V for
> 3.5 hours. I also want a crisp band because I am planning to cut
> these fragments out and extract the DNA for sequencing. Any suggestions?
>

> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
> _______________________________________________
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> Met...@net.bio.net
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[Michael Mattocks]

A 10% non-denaturing polyacrylamide gel will probably do the trick. If
you're familiar with SDS-PAGE this isn't much different, leave out the
SDS and run in 1x TBE (or .5x TBE if you prefer). Everything else in
terms of loading dye, markers and so on you can keep the same. It's a
bit easier to overload the gel so try a modest amount of product first.
You can extract the band for sequencing with an ammonium acetate
crush-and-soak protocol.

Mike

> Message: 1
> Date: Fri, 04 Dec 2009 15:17:08 -0600
> From: umwo...@cc.umanitoba.ca
> Subject: Separating PCR products on agarose gel with similar bp length
> To: met...@magpie.bio.indiana.edu
> Message-ID: <20091204151708....@webware.cc.umanitoba.ca>
> Content-Type: text/plain; charset=ISO-8859-1; DelSp="Yes";
> format="flowed"

[Dr Omar Hamarsheh]

I recommend to use 3.5-4% MetaPhor agarose gel (take care while preparing
the concentrated gel), run it at 150 volts for at least 4 hours. you'll
seperate these two small bands. we used it previously to characterize
fragments of microsatellites

good luck

On Fri, Dec 4, 2009 at 11:17 PM, <umwo...@cc.umanitoba.ca> wrote:

> Hi,
>
> I am trying to separate 745bp, 646bp, and 655bp fragments on an agarose
> gel. I've separated the 745bp fragment but the other two fragments are not
> separating on a 2.5% agarose gel, running at 55V for 3.5 hours. I also want
> a crisp band because I am planning to cut these fragments out and extract
> the DNA for sequencing. Any suggestions?
>
>

> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>

--
-------------------------------------
Omar Hamarsheh, MSc., PhD
Al-Quds University
Department of Biological Sciences
P.O.Box 51000
Jerusalem
Via ISRAEL

Tel: 00972-599197097
Fax: 00972-22796960

Email: ohama...@gmail.com

--
-------------------------------------
Omar Hamarsheh, MSc., PhD
Al-Quds University
Department of Biological Sciences
P.O.Box 51000
Jerusalem
Via ISRAEL

Tel: 00972-599197097
Fax: 00972-22796960

Email: ohama...@gmail.com