De novo sequencing of complex genomes is one of the main challenges for 
researchers seeking high-quality reference sequences. Many de novo assemblies 
are based on short reads, producing fragmented genome sequences. 
Third-generation sequencing, with read lengths >10 kb, will improve the assembly 
of complex genomes, but these techniques require high-molecular-weight genomic 
DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments 
for short-read sequencing are not suitable for this purpose. Methods of 
preparing gDNA for bacterial artificial chromosome (BAC) libraries could be 
adapted, but these approaches are time-consuming, and commercial kits for these 
methods are expensive. Here, we present a protocol for rapid, inexpensive 
extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our 
technique was validated using sunflower leaf samples, producing a mean read 
length of 12.6 kb and a maximum read length of 80 kb.