Ultralow v2

Example 4

Intact genomic DNA was fragmented in a 15 ul reaction containing 2 mU HL-dsDNase (ArcticZymes), 6 U E. coli DNA Polymerase I (NEB), 1.5 U T4 DNA Polymerase, 1×NEBuffer 2 (NEB) and 0.2 mM dNTPs under the following temperature profile: 25 C for 15 min, 65 C for 15 min, 4 C hold. The NuGEN Ultralow v2 ligation and PCR components were used to perform ligation and PCR as follows. Ligation was performed by adding 3 ul of Ligation Adaptor Mix, 5 ul of Ligation Buffer Mix, and 2 ul of Ligation Enzyme Mix for a total of 25 ul. After the standard ligation incubation steps of 25 C for 30 min, 70 C for 10 min, and 4 C hold, PCR components were added directly to the ligation reaction, without bead purification. 25.5 ul of Amp Buffer Mix, 2.5 ul of Amp Primer Mix, 2 ul of Amp Enzyme Mix, and 45 ul of water were added to prepare a 100 ul PCR reaction. After 9 cycles of PCR following the cycling conditions described in the Ultralow user guide the PCR products were bead purified and analyzed by Bioanalyzer.

FIG. 11 shows Bioanalyzer traces of library prepared by adapter-ligation and amplification of enzymatically fragmented nucleic acid. The resulting Bioanalyzer traces display normal looking libraries of the expected size and yield, with no evidence of adaptor artifacts, demonstrating the compatibility of no post-ligation purification with enzymatic fragmentation.