EMSA troubles

[Dutchdodo]

Hi everybody

i am doing the EMSA assay which made me mad, i used the Cy5 labeled primers to amplify the fragment for probe,PCR product was run on the agarose to confirm the unique PCR product. 1ul PCR product(with out gel purification) was used for EMSA, i always got additional band above the free probe, when i added less 0.5 ul PCR for EMSA, this additional band was less as well, do how can i address this problem? is it nessary to purify the PCR product before EMSA?

PS: lane1 and 4: only probe without protein
lande2 and 5: 0.025ug protein + 1ul and 0.5ul probe respectively
lane3 and 6 : 1ug protein + 1ul and 0.5ul probe respectively

waiting for your nice suggestions!

Best wishes,
dodo

[admin]

Hi there, seems you are getting some streaking and gel issues. What running temperature and buffer (TAE vs TBE) are you using? What current/voltage?

Also seems you have a lot of signal, how much probe are you running on there?

[Dutchdodo]

Hi there, seems you are getting some streaking and gel issues. What running temperature and buffer (TAE vs TBE) are you using? What current/voltage?

Also seems you have a lot of signal, how much probe are you running on there?[/quote]



Hi ,Thanks for your reply, i used the 0.5 TBE as running buffer at cold room 4 degree, and set the voltage at 120V, i Used 1ul and 0.5ul PCR products(50ul volume reaction ) for EMSA, but i did not purify the PCR products.

Xiaorong

[wr_1a]

thanks for u