This protocol describes a method for DNA extraction from E. coli, as an example of a Gram-negative bacteria. We recommend growing a culture to OD ~0.7, and extracting the DNA using the QIAGEN Genomic-tip 500/G columns. These are easy to use, do not require the use of phenol or chloroform, and give a consistently high yield. We have found that incubating the culture with chloramphenicol for 1 hour before collection resulted in a 4.5X increase in yield (from 7.4 µg to 33.9 µg). Addition of this antibiotic stops cell division, but still enables completion of DNA replication.

• QIAGEN Genomic-tip 500/G
• Chloramphenicol
• 10 ml renewed E. coli culture

1. Culture the E. coli cells until the OD reaches 0.5.

2. Add chloramphenicol to the culture to a final concentration of 180 µg/ml. Incubate the culture for another hour (at the end, the OD should be ~0.7). Remove 10 ml of culture for extraction.

3. Follow the QIAGEN Genomic-tip 500/G protocol. Elute the purified DNA into 200 μl TE buffer, pH 8, and leave at room temperature until all DNA has dissolved.

4. Critical Step: If after several hours some precipitation is still visible in the sample, this could indicate protein contamination. In this case, spin down the sample in a microfuge for 1 min at 10,000 g, and carefully remove the supernatant.

• Yield: 33.9 µg
• OD 260/280: 1.89
• OD 260/230: 2.29

- **Fragment size: 1.3 - 165kb (FEMTO pulse)**

Libraries for Nanopore sequencing were prepared using the Ligation Sequencing Kit, with g-TUBE fragmentation.

• Read length profile after fragmentation and sequencing: