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EMSA - probe design and competition


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6 replies to this topic

#1 lizbit

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Posted 12 July 2010 - 07:43 AM

Hi,

The lab that I am working in is really new to EMSA and functional genomics as a whole. Part of my thesis project is to design an EMSA study for a particular SNP in the TAGAP gene. I've got protocols for EMSA both radiolabelled and non-radiolabelled. I'm having trouble finding advice on how to design my probes.

How long should my probe be? Is this dependent on how big the presumed transcription factor is?

Also - how do I design competitor probes? How many competitor probes do I need? Is there a protocol somewhere that will tell me this?

I appreciate any and all advice.

Liz

#2 pcrman

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Posted 12 July 2010 - 06:50 PM

You can look into the literature to find out how people do such experiment. I actually did similary EMSA to study a snp. Regarding probe size, something around 20 nt is good enough. competitor is an oligo with unrelated sequence. One is good enough, of course the more the better.

#3 epibio

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Posted 13 July 2010 - 08:22 AM

I used to do competition EMSA with both "specific" competitor (unlabeled probe that is identical to your labelled probe) and "nonspecific" competitor (as pcrman described). In addition, you can use a series of unlabeled competitor oligos with different mutations in the putative binding site, if you're interested in that level of detail.

#4 lizbit

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Posted 13 July 2010 - 09:31 AM

I used to do competition EMSA with both "specific" competitor (unlabeled probe that is identical to your labelled probe) and "nonspecific" competitor (as pcrman described). In addition, you can use a series of unlabeled competitor oligos with different mutations in the putative binding site, if you're interested in that level of detail.


So does the sequences for the nonspecific competitor need to include anything specific (like a TATA sequence or a certain percentage of GC)- or is it just random sequence?

#5 epibio

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Posted 14 July 2010 - 07:03 AM

So does the sequences for the nonspecific competitor need to include anything specific (like a TATA sequence or a certain percentage of GC)- or is it just random sequence?


It doesn't have to, it can be random. In my case, I used sequences that contained binding sites for other, unrelated transcription factors, simply because I had them already in the lab.

#6 lizbit

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Posted 14 July 2010 - 08:34 AM

Thank you both so much! You've been a huge help!

Liz

#7 sata

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Posted 18 November 2010 - 10:18 AM

I am doing EMSA first time.Can you please tell me how to anneal the oligos? I am using 50 mM tris,10 mM NaCl & 1mM EDTA and then heat for 5 min at 95C & cool slowly on heating block.But it doesnt work I cant sea annealed probes.Can you plz tell me the procedure for annealing?

Sata




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