Why does my IVT gRNA product gives HMW smear in gel?

02 July 2024 0 6K Report

We are using MEGAshortscript™ T7 Transcription Kit for transcribing gRNAs from linearized plasmid templates. (MSP3485 digested with HindIII) (https://www.addgene.org/140082/) We let the reaction run for 4 hours at RT with 1 ug of our linearized template plasmid. Please see the attached gel result. The rightmost lane is the positive control that gives the correct 120 bases. My samples give large smear at a high molecular weight. What is causing this? The ladder is Thermo RNA RiboRuler high range. Our expected gRNA band is also around 120 bases. Thanks.

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