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1/5 Nextera Flex number of cycles
Hi,
We are about to start a fairly large experiment with the Nextera Flex kit in a very limited time. After some testing and reading experiences we have decided to do the kit at 1/5 strength. Now we are wondering if the DNA input should be adjusted as well. We will use 6uL DNA input (1/5 of 30), but does this mean that should contain >20ng (1/5 of 100) or still >100 to reach normalization with 5 cycles? Do you add a couple of cycles and if so how many? Is there such a thing as too many cycles? (we've been advised to do 16 cycles but that sounds like way too many but what about 8 or 10?).
I'm confident we will have enough library for sequencing but because of the number of samples that we plan to pool I am really hoping to use the kit's normalization and to avoid normalization by dilution.
We are about to start a fairly large experiment with the Nextera Flex kit in a very limited time. After some testing and reading experiences we have decided to do the kit at 1/5 strength. Now we are wondering if the DNA input should be adjusted as well. We will use 6uL DNA input (1/5 of 30), but does this mean that should contain >20ng (1/5 of 100) or still >100 to reach normalization with 5 cycles? Do you add a couple of cycles and if so how many? Is there such a thing as too many cycles? (we've been advised to do 16 cycles but that sounds like way too many but what about 8 or 10?).
I'm confident we will have enough library for sequencing but because of the number of samples that we plan to pool I am really hoping to use the kit's normalization and to avoid normalization by dilution.
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