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EMSA probes


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#1 DRP

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Posted 17 March 2010 - 10:55 AM

Hi,

I am new at EMSA and wanted some help with deciding labeling of probes.
My region of interest is pretty long ~230bp, so I want to make my own probe.
Also there are many binding sites spread through this region so I cannot make shorter probes, also I do not know what is binding to this region.
So, my question is if I can make this probe using a plasmid template with my region in it and oligos labelled with a fluorescent molecule like FAM, or IR dyes ? so at the end I will have a probe which will be a dsDNA with FAM at 5' ends of both strands. Will this work ? I was planing to gel purify this band and use it for my EMSA reaction with nuclear extracts. Has anyone used or read about using such probes ?
Any help will be greatly appreciated.
Thanks !

#2 pcrman

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Posted 17 March 2010 - 07:59 PM

For EMSA, a probe of 230 bp is too long and there will be a lot of non specific binding. If you really want to look into that whole area, go for footprinting using PCR product as probe. Once you pinpoint the binding site (protected area) by footprinting, then synthesize short oligos and use them as probe for EMSA.

#3 DRP

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Posted 18 March 2010 - 05:59 AM

For EMSA, a probe of 230 bp is too long and there will be a lot of non specific binding. If you really want to look into that whole area, go for footprinting using PCR product as probe. Once you pinpoint the binding site (protected area) by footprinting, then synthesize short oligos and use them as probe for EMSA.



Thanks for the reply. Yes, I did look into footprinting, and that is something I will have to standardize in my lab as nobody here has done it. Also it will be a fluorescent DNA footprinting since we cannot use radio isotopes.
However, EMSA of this 230 bp region was just to show the binding capacity of TFs to this region, so I was working on this first.

#4 Meral

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Posted 21 April 2010 - 10:58 AM

For EMSA, a probe of 230 bp is too long and there will be a lot of non specific binding. If you really want to look into that whole area, go for footprinting using PCR product as probe. Once you pinpoint the binding site (protected area) by footprinting, then synthesize short oligos and use them as probe for EMSA.


I have the same work with long RNA , could you please give me some links to footprinting??

Edited by Meral, 21 April 2010 - 11:02 AM.





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