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EMSA two fragment bands

EMSA two fragment bands - Protocols and Methods Forum

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Old 11-12-2009, 11:19 PM
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Default EMSA two fragment bands



I've been doing EMSA's with FAM labeled DNA successfully but we recently switched over to an Odyssey detection system using IR Dye which is much more sensitive and I have run into a problem. I use a labeled primer to PCR up my labeled fragment, I use one labeled primer and one unlabeled. I now see two bands in my no protein lane. I think the upper band is single stranded DNA. I know it's not contamination because I have several different fragments with different primers and templates and its shows up in them all. To get rid of it I've increased the amount of the unlabeled primer and reduced the cycles in the PCR reaction. This has not worked, even with 15 fold unlabeled primer. Does anyone have any ideas? Thank you so much!
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