FAQ: I don’t have a qPCR machine. Can I still use the NEBNext Immune Sequencing Kit (Human) (NEB #E6320)?

The qPCR step is optional; it’s only used to identify the optimal final PCR cycling conditions, as each sample can contain varying amounts of amplifiable molecules. One can achieve a similar result by manually taking an aliquot of the PCR reaction every 5 cycles and visualizing each aliquot by diluting it 5-fold and loading on an Agilent Bioanalyzer® or TapeStation™ to identify the optimal cycling number that yields the highest product with the lowest background and overamplification. Typically, most samples will amplify best in the range of 6-18 cycles, depending on RNA input.