I ran RNA denaturing gel (1.2% agarose, 1X HEPES-EDTA buffer and 0.45M formaldehyde). Used 0.5 ug of RNA, 5 ul of loading dye (HEPES-EDTA, bromophenol blue) and 1ul of Etbr in the reaction. After addition the tubes were incubated at 70 C for 5 mins.
Shouldn't all the bands be at the same position? I don't think the RNA has degraded because we can see distinct 28S and 18S bands as compared to RNA ladder.
What may have caused this different size of band?
Hi Nirjal,
Indeed it doesn't look like your samples are degraded. It looks as though, however, you have slightly faster migration in lanes 4 and 6 (from left to right). Because it is so lane-specific, I don't think there is a problem with the gel itself (i.e. uneven polymerization) or the run (i.e. unsteady voltage), but there is probably something different with the samples themselves. Are all samples in the same buffer? Are you sure the same amounts of EtBr and/or tracking dye were added to each sample? If not, the sample pH could change, which might affect conformation and thus migration speeds.
Thank you Lena for response. Yes, all the samples are in the same buffer. And I prepared a master mix of loading dye and Etbr before adding them in the tubes, so they should get equal amount of Etbr and loading dye.
Were the mice species considered obtained from diverse locations, regions or environments?
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