General:This is an optimized DNA isolation protocol adapted to the properties of bacteria isolated from biogas substrate and digestate, using NucleoBond\u00ae AXG Columns and NucleoBond\u00ae Buffer Set. The protocol reliably retrieves DNA of sufficient quality, length and yield. The performance has been validated on seven bacterial isolates from one biogas reactor and one biogas plant. Buffers and proteinase K can be found in NucleoBond Buffer Set III.Observed Performance on seven isolates runs using this protocol:N50 Ranges between 22,000 and 46,000 bp. General throughput is between 16-22 Gigabases of a single flowcell (flowcell 106 Rev D, using the LSK-109 Kit). Funding:This work was funded by the China Scholarship Council (CSC) [Grant No. 201606350190] and SLU.