I am still trying to wrap my head around using Nextera kits to get the maximum quantity and quality of sequence data from a MiSeq loaded with a pool of three yeast genomic DNA libraries (12 Mb). We have obtained acceptable data after three runs, but it has been hard to control the size of the library fragments.
Is it true that once one of the transposomes in a tagmentation reaction inserts its adapters into the target DNA, the unloaded transposase cannot cause any further mischief? If so, then knowing the number of active transposomes in the 5 ul of TDE1 or ATM should allow one to calculate the amount of DNA of a given size to combine with the 5 ul TDE1 or ATM to achieve the desired size distribution once the reaction has gone to completion. It is after all a poisson process.
Allowing tagmentation to go to completion would get around the variable results of a timed reaction. What is the flaw in this logic?
Is it true that once one of the transposomes in a tagmentation reaction inserts its adapters into the target DNA, the unloaded transposase cannot cause any further mischief? If so, then knowing the number of active transposomes in the 5 ul of TDE1 or ATM should allow one to calculate the amount of DNA of a given size to combine with the 5 ul TDE1 or ATM to achieve the desired size distribution once the reaction has gone to completion. It is after all a poisson process.
Allowing tagmentation to go to completion would get around the variable results of a timed reaction. What is the flaw in this logic?
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