Hey folks, so I'm trying to make some probes for ISH, and I'm using the Roche DIG Labeling Kit. I cloned my probe into a TOPO vector, linearized to leave a 5' overhang, and then did the T7 rxn as according to the kit directions. I did the optional DNase treatment as well. I then used the Qiagen RNeasy Mini Kit to clean up the RNA, and on the nanodrop I had a conc. of 335ng/ul (about 10ug total), and a ratio of 2.17. To make sure I ran 1ul out on a gel, and I know it's not ideal but I just ran a 1% TBE agarose gel w/ EtBr in it and I didn't heat my samples before loading the gel. I know the RNA doesn't run the same as my DNA ladder should, but I see two distinct bands in the RNA lanes. I'm wondering why there would be two bands after the in vitro transcription. Do they maybe correspond to denatured RNA and RNA w/ 2ndary structures? Thanks for any help.
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