When preparing plasmid samples we do RNAse treatment to remove RNA from plasmid DNA. I would like to know; is it necessary for plasmid sequencing from sanger di deoxy method ?
How RNA interfere with the sequencing proceedure?
Hi
It is ideal to remove RNA for sequencing. In fact RNA even interfere during regular PCR amplifications.
Hi Ajay,
I'm sorry I never experienced RNA interference in PCR so far. So I wondered what is the exact reason for RNAse treatment for sequencing. Could you please explain more. Removal of RNA, is it a must for sequencing?
Hi Nilmini,
Excess RNA do affect quality and quantity of PCR and there are several reasons for it. In plasmid, the contaminating RNA amount is in good amount. It can affect right from the precise quantification while making sequencing reactions to interfering in the functioning of Polymerase. It is total RNA from the bacterial cells containing all types of sequences, including those encoded by the plasmid itself. In addition, the RNA molecules can pair with the sequenced strand as well as with the growing strand , the RNA can form duplex regions and certain amount of polymerase may bind to it, smaller fragments may also serve as primers at multiple sites giving you a mixed signal etc., for there can be many reasons...for the RNA to interfere with the sequencing reaction..better to keep the preparation as clean as possible.
best regards
Dear Ajay,
Now I could understand why my genomic DNA with RNA didn't interfere my results as well. Thank you so much for you valued answer it explained all I need.
Hi Nilmini,
You are correct in pointing our than RNA in genomic DNA preparations can also be problematic..this is why we need to remove the non-essential contaminants from the desired component in most assays/analysis
best regards
Dear nilmini,
RNA presence in plasmid do interfere during sequencing as it's presence miss lead sequencing signals. Also RNAase treatment during plasmid isolation is important if you go for restriction digestion of recombinant plasmid and your fragment size is small than it will be difficult to see the fragment in the gel due to presence of RNA.
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