What can I do if my libraries are undercycled ?

In many cases, undercycled libraries will still contain sufficient amounts of cDNA for pooling and sequencing and will not need to be reamplified.

To check whether your library amounts are sufficient for sequencing, you can use our Lexogen's Library Quantification Calculation File.

How to use the Library Quantification Calculation File?

1- Enter the average library size of each sample (determined by a High Sensitivity microcapillary electrophoresis assay such as Bioanalyzer, TapeStation or Fragment analyzer device).

2- Enter the measured concentration (ng/µl) assessed by Qubit RNA assay.

3- Adjust the amount of each library to be pooled (minimum = 10 fmol).

The calculator will determine the volume of each library to pipet for pooling. As long as the volume indicated for pooling is below the volume of cDNA available, the library can be sequenced.

Should amounts of undercycled libraries be insufficient for sequencing, the PCR Add-on and Reamplification Kit V2 (Cat. No. 208) includes a Reamplification Primer that can be used to add some PCR cycles for your undercycled libraries (PCR Add-on and Reamplification Kit V2 User Guide, Section 6.1).