EMSA turned on me! Why? - I have been faithfully giving Diet Coke and kitkats! (Mar/19/2007 )

Seriously, I have been getting some great results with my EMSAs I just had some splotchiness on the blot so I redid "one more time" now the blot is clean but my binding changed!?! (the "negative control" mutated sequence looks like it has specific binding all the sudden-I did like 4 other times with "correct" result) Maybe I am missing something with the probe storage or too many freeze thaw cycles? I annealed one time 100ul volume 10ng/ul concentration using thermocycler, it is unusually warm in my lab right now, but not so warm that i would think the annealed primers would seperate but maybe i am wrong?

Everything else should be the same. Nuclear extract was made and aliquotted and stored at -20C no freeze thaws. Possibly significant difference in the incubation time (both with polydIdC and with probe, not more than doubled the time -- ie: usually 15 min dIdC and 30 min probe this time 30 min and 45 min) as I setup two gels worth instead of one.

Any suggestions?

are you using the same batch of nuclear extracts? I was just having this discussion with a labmate...

the 1/2 life of nuclear extracts is pretty short. if you're still using my protocol, then I would recommend storing the aliquoted extracts at -80, and assume they're worthless after about 3 or 4 weeks...I generally tried to use them up within 2 weeks, with no additional freeze thaws, if possible. for my needs, this wasn't a big problem; everything we did was within a short time-frame. however, if I needed my extracts to last longer, I would probably begin troubleshooting by adding more protease to the extraction buffers, perhaps?

I don't think the incubation time is your problem, although it's possible...I generally got better/stronger bands at longer times, and might miss the shift at too short. the only thing I can think of there, is if you're incubating at a warmer temperature perhaps you're seeing some degradation? do you do the incubations on ice?

I bet it is the extracts, I am still using the same batch--assumed they were ok because I was using aliquots from -80C. Easy to make fresh b/c I am using cell lines right now. I am setting up reactions on ice but incubation is at room temperature for 30 min-maybe warmer than usual temperature in the lab contributed to making degradation worse because of this. (I love spring but it seems like the facilities people cant keep up with the weather - have the heat on when its 70F outside and the AC on when it is 40F ) Will try again with new extract and let you know. Thank you for the help!

I'm just glad someone else is using it and getting it to work