If your libraries are undercycled or the yields are insufficient for pooling and sequencing, you can re-amplify your libraries using the PCR Add-on and Reamplification Kit V2. To check whether your library amounts are sufficient for pooling and sequencing, you can use Lexogen's Library Quantification Calculation File (found here).
For libraries with low but measurable yields, we recommend using 3-6 cycles for the library reamplification (approximatedoubling of the original yield per cycle). To be on the safe side, you can always use only half of your purified libraries for reamplification, and add one extra cycle more than you would use for the whole library as template. This way if your reamplified libraries are at all overcycled, you have the option to repeat the reamplification PCR with less cycles.
For libraries with no measurable concentration and/or no visibletraces on microcapillary devices (e.g., Bioanalyzer), we recommend using 6-10 cycles for reamplification. For further PCR cycle recommendations for reamplification, please contact: support@lexogen.com.
NOTE: We recommend performing quality control of finished libraries using 1) high sensitivity microcapillary electrophoresis (e.g., Bioanalyzer) for assessing library size distribution/profile and 2) Qubit high sensitivity assay for quantification.
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