HomeFAQsWhy is the yield for my transcription reaction lower than expected, and/or the bands are smeary?
FAQ: Why is the yield for my transcription reaction lower than expected, and/or the bands are smeary?
Be sure to carry out a control pilot reaction with the StuI Linearized Litmus 28iMal Control Plasmid. If the yield from your plasmid is substantially lower than that from the control reaction, it is possible your plasmid has RNase contamination. This problem can be solved either by further purifying your plasmid DNA, carrying out phenol extraction and ethanol precipitation steps. If yields from reactions containing your plasmid and the control plasmid are equally low, double-check the concentration of your template DNA, make sure your gel loading buffer does not contain SDS, and confirm that both the gel and tank buffer were prepared using ultrapure water (Milli-Q or equivalent). It is also possible that the linearization method used (restriction digestion or PCR) introduced RNase into both templates; again, phenol extraction followed by ethanol precipitation prior to transcription should solve this problem.
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