I am using PCR product as my template for in vitro transcription and then loading the Rna samples on 8M urea gel. I am observing multiple bands of both low and high molecular weight(my intended product is of 550bp). What could be the reason. Any suggestions?
did you purified the PCR product? you can get transcription from the PCR primers if they form dimers. The highest transcription can be from double stranded RNA made by the T7 when using blunt or 3' protruding ends. you can also have T7 termination signal (stretchs of U) in the template...
Didier Poncet Yes, I used a PCR-purified product for in vitro transcription (IVT). The concentration and purity of the PCR template are both good. I do have a poly U stretch at the end, which is intended to serve as the transcription termination point. However, I am seeing various smaller fragments, all under 500 bp. My intended product is 550 bp, but I am observing multiple bands, including those of lower and higher molecular weights.
the longer one can be missed termination (the polymerase pass throu the polyU... did you try longer incubation time? , higher temperature ? (T7 pol works well at 41°C and this can avoid the formation of secondary structure on RNA. may be reduced quantity of the DNA template (if you titrate the nucleotide with the polyU )... there is also special kits to transcribe short RNA (MEGAshortscript™ T7 thermo fisher)
Didier Poncet I am using the MEGAshortscript™ T7 kit from Thermo Fisher. I typically incubate the reaction at 37°C for 4 hours. I've tried lowering the temperature, but the issue of multiple transcripts persists. I haven't tested increasing the temperature yet, so I plan to try 42°C next. Extending the reaction time also hasn't resolved the problem.
in thermo fisher troubleshootings they suggest "Lowering the temperature to ~16°C or even 4°C can sometimes improve transcription. It is believed that lower reaction temperatures slow the polymerase's progression, thereby preventing it from being displaced by secondary structure or a string of one specific nucleotide." They also suggest to use another polymerase T3 or SP6... (but I am not sure that T3 or SP6 are better than T7)
Didier Poncet I tried lowering the temperature to 4°C, but the yield was very poor, and it wasn't sufficient to load onto the denaturing acrylamide gel.
- Badges
- Science method