How to quantify and what is the expected yield for Illumina DNA PCR Free libraries?
What is the expected yield?
Yield is usually at least 2.8 nM by single-stranded DNA (ssDNA) Qubit for libraries prepared with >300 ng input and at least 0.75 nM by KAPA qPCR for libraries prepared with 100-299 ng input or when using the low input protocol.
What is the best method for quantifying libraries?
Both ssDNA Qubit and qPCR are suitable methods for library quantification, with further specific guidance found in the Reference Guide. ssQubit is recommended for standard DNA inputs (>300 ng); whereas, qPCR is recommended for low DNA input (<300 ng). The conversion calculations for both methods will provide the double-stranded (ds) DNA molarity equivalent. Note that quantification with double-stranded DNA (dsDNA) Qubit reagents will result in quantification failure.
For molarity calculation purposes, 450 bp is used as the library size, 660 g/mol is used to adjust to the dsDNA molarity equivalent, and has been simplified to the equation below.
Note: if IPB size adjustment was made (per application note Tunable insert sizes with Illumina DNA PCR Free Prep, Tagmentation) to shift library size to 350 bp or 550 bp, then use 350 bp or 550 bp for the library sizing, respectively, and use 660 g/mol in the conversion equation to obtain the dsDNA molarity equivalent.
Why is there a difference between the qPCR and Qubit loading concentration? Both qPCR and Qubit ssDNA quantification are adjusted to reflect their dsDNA molarity equivalents. The remaining difference in loading concentrations is due to inherent differences in the quantification methods. The loading concentration for each method has been determined empirically.
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