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Non-specific bands on EMSA


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#1 marlinster

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Posted 09 June 2009 - 12:52 PM

Hi, I am looking for ideas....I have recently begun running EMSAs and have recently progressed from having no bands to acquiring bands that I cannot get rid of. Basically I lowered the amount of Salmon sperm DNA I have been using to 0.5ug in 20ul and got 3 lovely looking bands (one dark and two lighter ones). Unfortunately I get the same three bands regardless of which probe I use (and even get the same bands when I use a random probe) and now I am unsure how to proceed. I am trying to recreate results that were obtained by a previous lab member and so I know that these probes should work and give different results. I have tried a range of SS DNA (0.5ug was the lowest) amounts and find at the higher amounts a single band appears (same size as the dark one mentioned above) that gets fainter with more SS DNA.

I am not sure where my problems lie - I think my probes are annealing and labeling okay. Any ideas or suggestions would be gratefully received.
Thanks

#2 drjcroberts

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Posted 12 June 2009 - 11:17 AM

hi

need a bit more info. are you using DS DNA as your labeled probes? are they annealed oligos? PCr productss?

these sound like non-specific bands to me. you can also try dIdC or dGdC as your competitors. salmon sperm DNA can degrade over time and give odd results.

J

#3 marlinster

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Posted 15 June 2009 - 10:04 AM

Hi - happy to give you as much info as you need - I didn't want to bombard people with my whole protocol. I am using ds annealled oligos as my probe. I am almost 100% sure that these are non specific bands myself - I am just confused as to why I get the same non-specific band regardless of the probe used and even the size of the probe being used. Thanks for the info on salmon sperm DNA - I will buy some new stuff and try your other suggestions.

Thanks for giving this some thought,
Cheers,
Richard




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