i have the same problem

Dear fellow researchers,

I could provide an answer but I am not sure what you are trying to do. What are you trying to transfect for expression or reporter? Is it DNA you isolate or RNA (not clear from the question). If prepared from PCR it seems we are talking cDNA but then you use RNAseZap... Pls. vlearify and provide a picture and resp. conditions so we can help.

Best Boris

Hi Boris,

Sorry for the ambiguous description.

I am going to generate mRNA and encapsulate it into lipid nanoparticle. Therefore, I need a linearized DNA as a template and then perform in vitro transcription to get mRNA products. I PCR the linearized DNA from plasmid and purified it for further in vitro transcription.

This has also happened to me on the odd occasion, despite using RNaseZap and all RNase-free consumables, as well as changing gloves regularly. I don't think the issue is likely to lie with the DNA template if you have purified the PCR product appropriately. Consider using RNase inhibitor in your IVT reaction if you do not already. For purification, my solution is to not use the generic Zymo/Monarch spin kits (they don't get rid of all contaminants anyway), but to instead perform lithium chloride precipitation followed by cellulose spin column purification, and then isopropanol/NaOAc precipitation to get the final isolated mRNA product. If you want to buy all of these purification components in a single ready-made kit, a startup I co-founded sells mRNA purification kits here: https://www.messenger.bio/store/p/mrna-purification-kit. Hope this helps.