During the ligation step, the Ligation Master Mix and Ligation Enhancer can be mixed prior to setting up the reaction but ensure that the adaptor is not added to the master mix prior to setting up the reaction. Keep all reagents and buffers on ice unless otherwise noted and ensure the ligation reaction is set up on ice. It is more common to see adaptor dimers at low inputs. If adaptor dimer is present, bring volume of libraries to 50 μl with 0.1x TE buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead cleanup steps. Alternatively, especially for precious and low input material, samples can be pooled and second cleanup performed with 0.9X volume of beads prior to loading the sequencing instrument.
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