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DNA probe labeling for EMSA; How?


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#1 i_George

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Posted 05 July 2011 - 11:59 PM

Dear friends,

I try to do EMSA to check if my transcription factor of interest can bind with the promoter of my gene.
For the transcription factor, I express it in E.coli as a GST-fusion protein.

I have cloned the promoter of my gene and get some potential binding elements for the transcription factor.
Now, I plan to re-amplify the promoter in small fragments (180-300 bp)to use them as probes in EMSA.
In my lab, we used to use Biotin 3' end DNA labeling kit from PIERCE for single stranded-oligonucleotide probe.
In my case, they are double stranded and long probes and I guess this kit might not work ????

So I need you advice.
Do you guys have some kits that work well with my case?
or some other ways that can help?
(We cannot use radioactive labeling in my lab)

I hope I can get advice from you. Please help!!!

#2 phage434

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Posted 06 July 2011 - 03:44 AM

Do PCR with a 5' labeled oligo primer. Use either biotin or digitonin. Roche sells DIG detection kits. Another, possibly easier, approach is to spike DIG-dUTP into a PCR reaction to partially replace dT with DIG-dUTP in the PCR reaction. You can't use Pfu or other proof reading enzymes in your PCR, since they are inhibited by dUTP, but Taq works fine. Biotin works less well, in my opinion, but is certainly workable, and is much more cheaply and widely available.




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