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Old 03-20-2017, 10:26 AM   #2
cmbetts
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Location: Bay Area

Join Date: Jun 2012
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1) If I remember right, they mention in the paper that they got a statistically significant yield increase adding dNTPs at that step. It definitely would still work, just with somewhat lower efficiency if it's added w/ RT
2) Too much dT can generate artifacts, more dNTPs probably wouldn't have a noticeable effect. That same protocol can be used without modifying the RT conditions for bulk samples with >10ng RNA (only PCR cycles are reduced), so you don't need to worry about insufficient primer for any single cell applications. There are several orders of magnitude more primer than mRNA in the cell. You should do some preliminary characterization experiments with your cells to dial in the correct number of PCR cycles, so you might end up shaving off a cycle or two from the default if your cells are particularly active.
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