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What gel percent and Voltage is needed to separate 2680bp and 2600bp DNA nicely

agarose gel percentage voltage

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7 replies to this topic

#1 Inbox

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Posted 31 August 2012 - 05:22 AM

What agarose gel percent and Voltage is needed to separate 2680bp and 2600bp DNA nicely. So one can elute 2600bp without chances of contamination by 2680bp.

#2 gfischer

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Posted 31 August 2012 - 08:33 AM

I think you're going to find it very difficult to get complete separation of fragments that close in size.

Assuming this is a restriction enzyme digest, an easier solution would be to add another enzyme to cut the fragment you don't need into two smaller fragments, so you can more easily separate them from your band of interest.
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#3 hobglobin

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Posted 31 August 2012 - 09:08 AM

I'd agree with gfischer. But if you really have to do it:
High Resolution Agarose, 0,8 - 1%
Lithium borate or Lithium acetate buffer
running distance as long as possible
not long running times (buffers allow higher voltages), e.g. 20-30V/cm
In BioTechniques (2004) 37:598-602 (Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media) you find some more informations how to do it, though you might need to do some adaptation.
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#4 ascacioc

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Posted 31 August 2012 - 10:17 AM

I also think that gfischer's idea is the best; I don't see anything else to separate 80 bp apart...even the coolest running buffers in the world: the lithium borate and the lithium acetate

#5 Inbox

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Posted 31 August 2012 - 10:56 AM

Running long gel with high voltage, gel heat very much and if it is low melting agar it melts too. But it still unable to separate those bands.

Edited by prabhubct, 31 August 2012 - 10:59 AM.


#6 ascacioc

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Posted 31 August 2012 - 11:31 AM

yeah, but the Li based buffers are made for not getting heated at high voltages. If you don't have the Li salts for it, you can try the SB (sodium borate; need NaOH and boric acid) buffer from the same Biotechniques paper.

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Posted 04 September 2012 - 05:16 AM

Dear all,
Hi I just Digested 2680bp with restriction site present in it but not in 2600bp. This approach is Good. Thanks for other approaches too.

#8 ascacioc

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Posted 04 September 2012 - 05:32 AM

glad to hear that. Sometimes the easy-clever way is the best ;) Kudos to gfischer





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