Dear colleagues,
I'm currently running some in-vitro transcriptions with the HiScribe T7 RNA Synthesis Kit by NEB. As Templates I'm using a purified PCR product as a run-off template. I properly thought about my Promoter sequence, bearing all the necessary bases not only for the binding but also for the initiation site. I'm expecting a ssRNA of roughly ~750-780 nt for my transcripts. However, my Urea-PAGE looks like displayed here. I can't really explain all the additional bands...
After the in-vitro transcription I'm using a RNA clean up kit and purify my transcript prior loading on the gel. I don't think it's unspecific degradation, the band look to sharp for that.
I'm looking forward to your suggestions and expertis
Hi Moritz
did you test your primers to be sure they're specific enough to run properly your PCR and avoid smears as in your gel?
for example you can test an in silica PCR at UCSC (https://genome.ucsc.edu/cgi-bin/hgPcr) and see if you're on the best way.
good chance
fred
Frederic Lepretre, thank you for the fast reply. I took caution by the primer design and I assured the quality and specificity of the template pcr on agarose gel after pcr clean up. Attached also a picture of an agarose gel of 3 samples of eGFP (~779 bp).
PCR are very clean, ok.
one other solutions is the state of your samples. did you test QC on them (BIOanalyzer...)?
fred
quality control, just to be sure samples are not degraded too much that could disable the best amplification.
on the other hand, you can also use DMSO at 5% to enhance PCR
fred
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