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Is there an optimum % agarose or... "more is better"?

agarose separation resolution

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#1 assembler01

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Posted 30 January 2012 - 09:54 AM

I was under the impression that above a certain percent concentration of agarose in your gel (TAE electrophoresis of DNA) the larger bands won't resolve as well anymore. There are even websites out there that have graphed distance between bands at different percent agarose and come up with optimum % agarose for a particular band size. However, my PI and this other lab keep telling me to crank it up and use higher and higher concentrations of agarose to try to resolve close bands. So, if you have two bands that are close (like 890 and 930bp) do I trust the calculator and run a 0.9-1% agarose gel or do I run 2-3% gels and just run it for an extremely long time? Which gives better resolution assuming you run both to near the end of the gel?

Thank you.

#2 hobglobin

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Posted 30 January 2012 - 10:07 AM

I'd use the recommended agarose percentage and avoid long running times because with increasing time bands become more and more broader because of diffusion. Second you may try a different buffer such as lithium borate or sodium borate that both increase resolution a lot and can be used at higher voltages too (reducing running times).
BTW if you use ethidium bromide in the gel and use the whole gel, most likely you have to dye the gel afterwards again, because Etbr moves to the opposite direction of the DNA.
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#3 scolix

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Posted 31 January 2012 - 05:37 AM

You could run a higher percent gel but if they will resolve such small differences without doubt is difficult to conclude.

As suggested try sodium or lithium borate, they help.





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