Hello, I am currently trying to clone and express an ORF of interest with a known sequence that I obtained from a de-novo transcriptome assembly. However, because my original cDNA was sheared in the pre-transcriptome processing, I am unable to directly amplify the sequence of interest from these samples via PCR, pull down, etc. Since I know the full sequence of the complete ORF, would it be possibly to simply synthesize a duplex DNA strand and use this to clone? Or do I have to start over and make more cDNA? Thanks
I don't see why there would be a problem in synthesizing it, as long as you feel confident that the sequence is correct. However, you can probably go back to the RNA and remake the cDNA again and PCR the ORF. That way you can sequence it by Sanger to be sure the de-novo was correctly assembled.
Hi Madeleine, as Ron suggets you can indeed synthesize it, but it can get pricey depending on the size of the ORF. Instead of PCR from the cDNA, it would be easier to PCR from a genome prep if you have one made? Then you dont need to deal with fragile RNA.
Hope this helps, Andrew
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