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I see big fuzzy bands around 100bp as well. They're most likely RNA contamination. To get rid of them, digest your RT-PCR products with RNAse-H. But if you just need to visualize your band of interest, and the fuzzy bands aren't getting in the way, it shouldn't be a problem. I usually input anywhere from 1-2 ug of RNA into my RT-PCR reaction using the ...


4

There is no single "ideal amount" of RNA. I would suggest you to do a titration curve to determine the best amount for your specific assay. The band at 100 bp could be a non-specific amplification. You could try to increase slightly the annealing temp to see if that removes (or reduces) the lower band. Alternatively, you can consider a different primer set ...


2

The best way for scaling up these kinds of reactions is to set up many reactions. Prepare a mastermix for lets say 20 reactions. You can pool them together, precipitate the RNA and dissolve to appropriate concentration in nuclease free water. Also, use gene specific primer instead of oligo-dT and don't add poly-A tails (poly-A does not affect the in-vitro ...


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You are right, they should have written "all currently known PERVs". Although the structure of ERVs is generally well understood (https://www.ncbi.nlm.nih.gov/pubmed/26104559; https://www.ncbi.nlm.nih.gov/pubmed/26818261) there may be some complex cases that have been missed. Also, the map of the porcine genome is not fully complete yet (nor the human one) ...


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Interpreting the question Initially I interpreted the question as little more than “Why one type of virus rather than another?” (dsDNA. ssDNA, ssRNA [+ve v. –ve] etc.), and this (which would be unanswerable) was the way @Taimur seems to have understood it. And I also felt that the poster’s reference to ‘complexity’ reflected more his familiarity with other ...


2

I am going to focus the answer on mainly "why HIV virus has evolved such mechanisms to go from RNA to DNA and back to RNA when it could simply use the first RNA to make its copies". While others have already discussed the broad point, I will discuss more about the details. There are a few points which might support this, all of which basically come down to ...


1

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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Viruses are one of the particles whose evolutionary origin is still a mystery to scientists. Various hypotheses have been presented but none of them have been proven so far. However, as one of our commenters has said, "What works, works and what doesn't, doesn't." This is the basic concept of the evolutionary theory. All organisms which can survive in an ...


1

Nanodrop may be fine for the volumes but as you rightly guessed it is not so easy to distinguish DNA from RNA (and primers). Qubit (invitrogen) is a sensitive, dye based quantification assay. You can use that for estimating cDNA yield. The kit contains different dyes for DNA, RNA and protein estimation.


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