2 replies to this topic

[marlinster]

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

[drjcroberts]

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

[marlinster]

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