why am I getting to see 2 RNA bands (one expected and one extraneous) after performing In vitro transcription reaction with T7 RNA polymerase using HindIII linearised DNA as a template. Even after HindIII digestion, I observed only a single linearised DNA in the agarose gel which is then followed by its phenol/chloroform extraction. And then this gel extracted DNA was used to set up the in vitro reaction, but still I got to see 2 RNA bands. What could be the reasons?
It is common to observe two IVT RNA bands, both of which are correct and represent different secondary structures.
if the IVT RNA is used as a probe for hybridization tests such as Northern blot or in situ hybridization, it's acceptable, as it will not affect the results.
Hope it helps
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