Posted 17 March 2010 - 07:55 AM
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.
Posted 17 March 2010 - 03:15 PM
Posted 18 March 2010 - 06:03 AM
Try DIG labelled PCR of your probe sequence, should work fine.
Thanks for the reply. I have used DIG label for synthesizing probes for in-situs but DIG is something that incorporates within the sequence, and I am trying not to modify the sequence and just end label it.
My concern was if I end label with FAM is it efficient enough as compared to biotin or P32 labeling ? and I am still trying to find some literature on it.
Posted 18 March 2010 - 02:59 PM
Posted 18 March 2010 - 04:00 PM
You can end label with DIG too, so it should only affect a single base on one end of the probe. I don't know about FAM, but basically anything you use to label will interfere with the sequence somehow.
Oh...thanks, I will try that..will much cheaper for me too..since we have a ton on DIG in our freezer.