What are the recommended conditions for long-term storage of my libraries?
For long-term storage, libraries should be stored concentrated (dilutions recommended for template preparation are not intended for long term storage) in LoBind tubes in low TE in non-frost free freezer at -20 degrees C.
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If adapter dimers are present in my library, can they be detected using the Ion Library TaqMan Quantitation Kit (qPCR)?
No, the Ion Library TaqMan Quantitation Kit for qPCR library quantification is unable to differentiate amplifiable primer-dimers from library fragments. We recommend assessing the library size distribution, including checking for the presence of adapter dimers, using the Bioanalyzer instrument. Libraries containing adapter dimers will have sharp peaks at ~70 bp for non-barcoded libraries or ~90 bp for barcoded libraries.
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I tried quantifying my amplified library with both the Ion Library Quantitation Kit (qPCR) and the Bioanalyzer instrument. The concentration according to qPCR is lower than the Bioanalyzer instrument. Which concentration should I use?
We recommend using the qPCR result, as quantification by qPCR is generally more accurate than quantification using the Bioanalyzer instrument.
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Can the Ion Library Equalizer Kit (Cat. No. 4482298) be used to normalize 400-base read libraries?
Currently, the Ion Library Equalizer Kit is validated and only recommended for use with up to 300-base read libraries.
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For quantification of my library on the Bioanalyzer instrument, do I have to amplify my library first?
We highly recommend quantifying unamplified libraries using qPCR, as it specifically measures the amplifiable (or usable) library fragments present. Unamplified libraries will contain unadapted and improperly adapted fragments, which will be included in quantification data obtained if using the Bioanalyzer instrument. Library quantification on the Bioanalyzer instrument using the Agilent High Sensitivity DNA Kit is only recommended for amplified libraries, as the library amplification process selects for properly adapted library fragments.
The Bioanalyzer instrument can be used to assess the size distribution of both amplified and unamplified libraries. Please see the Ion Xpress Plus gDNA Fragment Library Preparation User Guide (Pub. No. 4471989) for detailed information. If using the Bioanalyzer instrument for quantification of unamplified libraries, please note that this method will generally underestimate the concentration of usable library fragments and a library input titration may be necessary to optimize template preparation.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.