I’m just curious about how the RNAs designed for the lab are synthesized. Are they made nucleotide by nucleotide? From DNA patterns? From oligonucleotides? Do you make them in bulk or make one and then copy it? Do you store the sequences as cDNAs? I’m sure there is some big machine with a proprietary procedure at work somewhere, but I’m just wondering generally.
We start out by ordering DNA oligonucleotides from a company who specializes in producing them cheaply. Then, we use PCR assembly to put together the short oligonucleotides to form a full DNA construct. This DNA construct serves as the template for our RNA. Once we have the DNA template, we can use a version of the enzyme RNA polymerase to transcribe our DNA and produce RNA. We use some of the RNA for our chemical mapping and frequently have plenty left over - we store it in freezers long term so that we can go back and revisit the same sample of RNA later if we want to gather more data on the entries for any reason.
You already mentioned a lot of these ideas - that’s great! Let me know if you still have more questions.
Thanks for the explanation. I guess it helps that many of the sequences are similar. Kind of like molecular legos. Thanks also for letting us all participate in this terrific project!