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First of all you need to figure out what your goal is: an expression vector (i.e. plasmid) which will drive expression of a gene in bacteria (let's say E.coli), and your gene's coding sequence needs to be downstream of the promoter in that construct. Your task is asking for a pretty routine lab task known as cloning (https://en.wikipedia.org/wiki/...


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It isn’t really clear exactly what the questioner is asking, but… ...one way of thinking about this is to ask how likely is it that a low-abundance RNA will be absent from the sample taken from the preparation of RNA. This would make the sample non-representative at the qualitative level. The question states that 12,000 ng of RNA is isolated. Since nothing ...


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To R! Download the most comprehensive GTF file from Gencode for mouse. Do the following in R (I'll put comments in the code so you can hopefully follow along): library(GenomicFeatures) #Load the GTF file and make a TxDb object txdb = makeTxDbFromGFF("gencode.vM13.annotation.gtf", format="gtf") #Make a GRangesList, with transcripts split per gene grl = ...


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You are correct about the top loop being the intronic region of the gene that would be spliced out of the mRNA that was used to make the cDNA and will not have complementarity to the sequence found in the cDNA. You are also correct that because it is cDNA, then it cannot be RNA for the bottom box. Remember that the cDNA will contain what the mature, ...


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Two answers follow: Technical experience of many: it should be no problem to have the same protocol with a twice-lower starting concentration, without making any adjustments whatsoever. The suggested 1ug is very much on the safer side of things, 2-fold fewer transcripts makes almost no difference. Maybe at 1 or 2 orders of magnitude (10 to 100s) less will ...


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