Hemo klentaq reaction buffer

Probe-captured miR samples were then amplified and indexed via polymerase chain reaction (PCR) using master mix (10 mM deoxyribonucleotide triphosphate (dNTP) 5× Hemo Klentaq® Reaction buffer, Hemo Klentaq® DNA polymerase (New England Biolabs Inc., Ipswich, MA, USA), forward, and reverse primers (HTG Molecular Diagnostics Inc., Tucson, AZ, USA)). The PCR conditions for the reactions were: (1) 95 °C for 4 min, (2) 95 °C for 15 s, (3) 56 °C for 45 s, (4) 68 °C for 45 s, (5) repeat steps 2–4 for 16 total cycles, (6) 68 °C for 10 min, and (7) hold at 4 °C. Following PCR, library cleanup was performed using Ampure^® XP beads (Beckman Coulter Inc., Brea, CA, USA). The samples were mixed and incubated with the beads at room temperature (RT) for 5 min. The bead-bound libraries were then washed twice with 80% ethanol to remove any leftover PCR reagents and enzymes. After washing, the samples were dried at RT for 2 min to allow the residual ethanol to evaporate. The libraries were then eluted off the beads with 10 mM Tris hydrochloride (Tris-HCl), pH 8.0.