Hi all,
I am performing an EMSA assay for RNA-Protein binding and I am using SYBR Safe dye. I have 0.75% agaros gel that contain 1X THE buffer and 100mM NaOAC. As you can see, I have lots of background noise in my gel. I was wondering maybe someone have a suggestion about how Ican get rid of it or decrease it.
I appreciate your help in advance.
Judging by the distribution of particles, it looks like one or more of your reagents is contaminated.
Does this happen when doing routine gel electrophorisis?
If so, use new buffer and agarose and/or DNA dye.
If you omit the NaOAC, do you get the same result?
If so, prepare it fresh. You could also try filtering it.
Good luck!
It looks like dust or other particulates. Try filtering your running buffer through a 0.45 micron filter before using it to cast and run the gel.
Or air bubbles. Try to bring your gel to boiling 3-4 times - that will degas it. Also, run the gel at lower voltage so that it doesn't heat up (or use a fan to cool it down while running). Do not keep the gel in a fridge - that tends to create the bubbles when running.
Thank you all. all my buffers were freshly filtered and in nuclease free water except running buffer (1x THE) that I would filter the stock when I make it. I decreased the concentration of salt. it helped a little. Filtering the running buffer before run was helpful as well. Yet, I see noises. I do not have lots of control on bubbles since I boil it everytime and I have to run the gel in 4 degree room. Do you think it is possible that THE Buffer has an effect?
Adam B Shapiro Ryan Wheeler Aidas Nasevicius
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