I want to inject fly embryos with a collection of 50 different RNAs that I would like to in vitro transcribe from the lowest number of plasmids. If I insert in the plasmid 5 sequences (or more if there is no limitation), leading the RNA of interest, each spaced by a transcriptional initiator and terminator, will I obtain an equal amount of the 5 corresponding RNAs? Will the RNA Polymerase be able to transcribe the first gene in the same way of the fifth one?
With this method instead of doing 50 in vitro transcriptions I’ll be able to do only 10.
In vitro transcription generally uses T7 or SP6 polymerases. These are run off transcripts, meaning that the transcription ends when it hits a double stranded break, usually a restriction site that you have cut your plasmid with. The RNAs are then capped and polyadenylated in a separate reaction to make viable mRNAs. The T7 promoter is a short sequence, but termination sequences are poorly defined. Thus, it is unrealistic to try and clone different coding regions into the same plasmids. I think you should reconsider your strategy. Also, ask yourself why you need to inject so many RNAs at once.
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