I know that this post is dead for 3 years now, but I had the same problem and I know that many people are looking for an answer. What they are telling me is that tagmentase is an extremely labile enzyme and it is enough for it to be a little bit outdated or to have several freeze-thaw cycles to lose most of its activity. Hence very large library sizes - tagmentation is just not good enough, and increasing its duration will not help because the enzyme is inactive. Better switch to other methods of DNA shearing.
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Eh? Who are the "they" to whom you refer? Nextera doesn't seem particularly labile to me.Originally posted by rudzik79 View Post
One point of possible confusion: after tagmentation, but before PCR, everything tends to look very big because the Tn5 has inserted but apparently it still tethering both DNA ends to each other. See here for more details:
This paper describes a "contiguity preserving transposition based sequencing" method that takes advantage of this behavior of Tn5.
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ReallyOriginally posted by rudzik79 View Post
Last posting in this thread was on 20th April of this year by @fanli
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Yes, I think it is possible that rudzik79 did not read the entire thread prior to posting. Which one could understand, I suppose. But in this case led to an erroneous assumption as to the "aliveness" of the thread...Originally posted by GenoMax View PostReally Last posting in this thread was on 20th April of this year by @fanli
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