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I would like to have pure single stranded cDNA of my choice. I am thinking of reverse transcription from RNA (obtained by in vitro transcription), degrading RNA from RNA-DNA hybrid with RNase-H, heating to 65C to remove any oligoribonucleotides still bound to DNA and pass the solution through G50 sephadex column buffered with elution buffer. This is what I have in mind but are there any efficient ways that are tried before? Or do you have any suggestions over this protocol?

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  • $\begingroup$ You need a DNA template to do in vitro transcription, so I'm not really understanding what you want to do here? How long is your sequence? $\endgroup$ – Mad Scientist Aug 14 '15 at 11:46
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    $\begingroup$ You need to use reverse transcriptase and a thermal cycler. Not much more to it, follow manufacturers suggestions. Life tech makes good RT $\endgroup$ – rhill45 Aug 15 '15 at 0:33
  • $\begingroup$ @MadScientist I think he's talking about rna->cDNA here not invitro Txn. $\endgroup$ – rhill45 Aug 15 '15 at 0:34
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Sure that will work, or gel purify the ss cDNA, or treat the rxn with alkali to degrade RNA. The real questions in my mind are:

  1. What will you use as a primer for the RT?
  2. What are you going to do with this pure ss cDNA?

If you already have the target cloned (how else would you transcribe it in vitro?), then why not just do a linear PCR where you cut the plasmid to linearize it at the site of your choosing, and then throw it in a PCR machine with just one primer?

Why bother going through the T7 RNA pol and RT steps (if you don't need to)?

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