The desired gene region was amplified by PCR and the resulting PCR products were cleaned. Bands were observed in an agarose gel and measured in a spectrophotometer. Then, transcription was performed with the ABm Onescribe T7 transcription kit (E081). Of the PCR products, 196 ng for gene 1 and 133 ng for gene 2 were included in the transcription reaction. Despite the assurance that the kit was functioning correctly, the transcription products did not yield bands in agarose gel electrophoresis. No bands were observed with or without the optional DNase treatment following transcription in the protocol.
1. What could be the cause of the transcription problem?
2. What are the alternative methods to prevent DNA loss in the clearance of PCR products despite increasing the initial concentration?
3: How can the formation of dsRNA after transcription be determined? Can it be visualised with agarose gel electrophoresis? does the PCR product and the transcription product give the same gel image? Can it be measured in a spectrophotometer and what is used as a blank?
İlayda NUR Belen
Please, look at discussion in https://www.researchgate.net/post/MRNA_synthesis_and_purification_T7_MEGAscript_Transcription_Kit_troubleshooting
There is one advice by @Victor G Stepanov that I would recommend too: "Actually, it might be useful to set up the T7 transcription reaction in small scale (~ 20-30 mcl), and then resolve the reaction mix on a gel to see if you had the mRNA synthesized in the first place."
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