In vitro transcribed sgRNA on agarose gel - can anyone help me identify these bands?

[ray.n...@gmail.com]

Hi everyone - so I have recently had success generating guides by in vitro transcription, using a template construction strategy based on the one described in this paper. However, when I test the activity on target DNA with Cas9 and run the result on a normal agarose gel, along with the expected fragments I see some blurry bands that aren't there when the reaction is done with commercially ordered RNA. I'm not sure whether this is RNA, or maybe leftover DNA from the IVT template (I used an RNA clean-up kit but no DNase treatment). What's peculiar is that the bands are different for the two guides - maybe secondary structure differences due to unique guide sequences?

I'm worried about whatever is there interfering with later transfection experiments.

Thanks for any insights you may have.

The lanes are:
1) 1 kb+ ladder
2) Guide 1, commercial
3) Guide 1, IVT
4) Guide 2, commercial
5) Guide 2, IVT
6) Guide 1, no target DNA (ie just RNA + Cas9)
7) Guide 2, no target DNA

Cleavage reaction was heated at 37 for 1 hour, then 65 for 10 minutes.

[Gita Reinitz]

In vitro transcription usually results in multiple transcript creation. The RNA polymerase can stall and end before reaching the end of your template, resulting in smaller products. You want the product that is the correct size for your expected RNA. A smear probably indicates many unwanted products. Commercially synthesized RNA, unlike invitro transcription, gives you only the product that you want

Hope this helps

Gita