Is library amplification required when using the Normalase™ Module?
Yes, libraries must be amplified and conditioned with Normalase™ primers. Even if library yields have met the 6 nM or 12 nM minimum threshold before PCR amplification, a minimum of 3 cycles is required to condition the library for downstream enzymology. The Normalase Module allows you to maintain your conventional library amplification reagents, except for any amplification primers.
For indexing by ligation, use Reagent R5 for Normalase PCR in place of conventional amplification primers and see Appendix: Section C in the protocol for recommended minimum PCR cycles.
For indexing by Normalase PCR, use Reagent R6 for Normalase CDA or Reagent R7 for Normalase UDI. Normalase indexing primers complete the adapter sequence as well as amplify and condition libraries for downstream Normalase steps. See Appendix: Section C for minimum cycling as the use of Normalase indexing primers requires more cycles compared to conventional indexing primers.
Note: You can refer to the xGen™ Normalase™ Module Protocol and the specific xGen™ library preparation protocols for minimum cycle recommendations.