Dear all, I am trying to set-up a RNA in situ hybridization experiment.
I am reading several protocols for that, and I found one paper says 'if in vitro transcription is performed well, you will see a single band on the gel'. And the other one says 'you will get multiple bands if in vitro transcription was successful'.
What is right? I am confused.
Waiting for your help. Thanks!
Ideal product shows distinct bands. Minimal band diffusion is acceptable. Sometimes you get two bands, due to secondary structures of the RNA. you can try a "quick and dirty" method. Heat the sample for 5 minutes at 65°C and then immediately cool it down in an icebath and keep it there until loading. By doing so, you melt up the secondary structure of the RNA and keep it in this state. You will most likely then see only one band.
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