Many liquid handlers do not have additional heat block positions on deck to pre-warm mastermixes. However, the most critical step for QuantSeq first strand cDNA synthesis is that the RNA / FS1 samples are kept continuously at 42 °C after denaturing (step 2), and while the FS2 / E1 mastermix is added.
Although pre-warming this mastermix helps to prevent mishybridization by maintaining the sample temperature of the reactions after denaturing, the pre-warming can be omitted for automated protocols as long as the FS2 / E1 mastermix is prepared in advance and kept at room temperature on deck. This
ATTENTION! The FS2 / E1 mastermix should not be cooled or kept on a refrigerated block on deck prior to adding to the samples (at step 3).