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Uneven bands denaturing agarose

RNA agarose

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#1 nprashu

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Posted 04 September 2012 - 11:27 AM

Hi All,
I have recently started doing denaturing northern blots for RNA using 2.2 M formaldehyde in a 1% agarose gel. UV exposure of rRNA using ethidium in the loading buffer shows uneven bands with more signal on the edges with streaks running above and below only on the sides. This is resulting in ugly blots and prevents any kind of quantitation. Here are the details below:

RNA amount: 20 ug with 200ng etbr in 20ul
Running conditions: 80V for a 15cm gel in 1X MOPS buffer pH 7.0 for 4 hrs at RT

Any advice on how to get sharp bands? Also, any suggestions to prevent cross hybridization to rRNA during isotopic probing with single stranded RNA probes is also welcome.

#2 bob1

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Posted 04 September 2012 - 01:10 PM

Try adding EtBr to the running buffer to get even staining of the gel.

#3 nprashu

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Posted 04 September 2012 - 04:03 PM

Unfortunately, I know that this is now a etBR staining problem because the bands appear uneven with streaks on the sides even when I probe with a radiolabelled RNA.





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