Hello!
Recently, I performed a in vitro transcription to try to get a 30kb RNA. Could someone tell me a good protocol to check the integrity of this large RNA??? I have thought about running the samples in non-denaturing agarose gel made with TBE 1X with SYBR safe stain and, before loading the RNA samples, mix them with formaldehyde loading dye and incubate them 65ºC for 15 min before loading the gel.
Running IVT RNA out on a gel is a pretty standard way of checking the efficiency of your IVT reaction. With a 30kb product, you're going to need a long gel to see the full 30kb product and any shorter transcripts produced by early termination.
John Hardy Lockhart long gels are a bit out of fashion but if the primers were dye labelled could a capillary sequencer be used with an appropriate end labelled size marker set
Paul Rutland , a capillary system might work better than a gel, but I don't know if ladders that go up to 30kb are available, but he might be able to get some idea using a TapeStation or something similar.
Ricardo Requena Platek , the massive size of your transcript is going to make it hard to resolve both your 30kb product and any small early termination products. In my experience we did not have any significant amounts of shortened transcripts when doing up to a 10kb RNA, but I've not tried transcribing anything larger than that.
Thank you for all your comments, I will take them into consideration. I will run a long gel tomorrow but if the result is not good I will try to get a capillary system.
By the way, one more question..... Which gel stain is the best for RNA, SYBR safe or GelRed?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA