I am doing an IVT of guide DNA sequences. My template DNA is ~120 bp, but after the T7 promoter, there are only ~100bp. But after I do the IVT and run on a gel, I get two separate bands. I am using a DNA ladder (instead of an RNA one) but according to that the two bands are ~200bp and ~130 bp. Some of the IVTs even have 4 or 5 different bands, even longer than 200bp.
**I have run my DNA template on a gel and it only shows one band around 120bp as I was expecting.
**I'm using the IVT kit from NEB
It is normal that single stranded RNA will form secondary structures, which will make it appear larger on a gel. You could try to denature some of the RNA at 70 degrees for 5 minutes before running it on the gel and see if this removes the extra bands.
Did you use HiScribe T7 high yield RNA synthesis kit? Can you solve your problems according to trouble shooting following user manue?
Deelaka, this is a common occurrence with T7 RNA polymerase. In the absence of T7 transcription stop sequence the polymerase will albeit with low frequency proceed to transcribe in a rolling circle fashion. You may see not just 200, but also a ladder with decreasing amounts of 300bp, 400bo and so on in you IVT reaction where the majority should be the 100 bp sgRNA.
DNA ladder is dsDNA, accounting to lower size of ssRNA. Secondly, I also think non-denaturing PAGE could show nucleic acids still bound to enzyme in different ratios resulting in two if not multiple bands upon sensitive staining. So, denaturing a small aliquot before agarose or PAGE must help. Thanks.
I had the same problem while using HiScribeTM T7 Quick High Yield RNA Synthesis Kit. The issue was incomplete digestion of DNA template, which yields in bigger bands than expected.
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