Originally posted by Simone78
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I'm trying hard to coerce as much efficiency and yield out of the protocol as possible, as I'm doing RT on a very low abundance transcript. Looking around different TSO protocols, it seems people have slightly different approach to the cycling condition of the TSO reaction: could you comment on the 72 degree annealing and the subsequent cooling step? some protocol go directly from the annealing to cooling to 42 degree, while the SMART-seq2 protocol specfiically says to cool it on ice. Have you seen what the difference is in terms of yield? How about the length of template annealing step? Do you think increase the time of primer annealing could help with low abundance transcript?
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