Dear all,

I am performing an in vitro transcription of 300 bp long ALU RNA, 600 bp long ALU RNA (two copies) and 900 bp long ALU RNA (3 copies). While 300 and 600 bp templates seamed to provide the desired products, 900 bp DNA template gives us either nothing or 600 bp long RNA products at best. We tried to sequence the DNA template and there was a problem with sequencing too, which hints there might be hairpin loops formed somewhere.

Is there a way how to test the presence of hairpins and how to overcome their formation while leaving the template suitable for the transcription? (I found that hairpins can be melted with high temperatures or that hydrogen bonds can be disrupted with Mg+ ions but I need to be able to at least return the conditions back to normal for the transcription while preventing hairpins from forming again).

Thank you for your help.