Any idea what these bright smears in my gel are? This is an in vitro transcription reaction, after using the Invitrogen™ MEGAclear™ Transcription Clean-Up Kit. I have used the kit many times in the past with no issues. The ladder is low range ssRNA, bright band is 300 bp, next up is 500 bp. – Kelly M.
Thanks for your question. To exclude the possibility that the smear is coming from MEGAclear kit, you can run a negative control (using H2O as input) and run the eluate on the same gel to check it. Also, to exclude contamination from the in vitro transcription kit, you can run a negative control reaction (using H2O as DNA template) and a positive control reaction (using the DNA template included in kit) to compare with your samples. If all of these controls do not show the same smear band, then one of the possibilities is that the smear band is part of the in vitro transcription products generated from the plasmid/vector.
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