DNA extraction is the first step of any sequencing project. It’s often considered the easiest part of the workflow, compared to the more complicated steps of library prep, sequencing, and data analysis. But don’t take it for granted—a few key considerations in your DNA extraction protocol can make all the difference for your sequencing data.
Here, we’ll explain our top tips for DNA extraction and how Nanobind extraction kits can help you make the most of your HiFi sequencing run.
Get ultra-clean, high-molecular weight DNA with Nanobind DNA extraction kits
Long-read sequencing with HiFi reads requires purified high-molecular-weight (HMW) DNA. Unlike short-read sequencing, which can more easily accommodate varying DNA quality, HiFi sequencing performs best with long molecules.
To get the HMW DNA you need for your HiFi run, we recommend Nanobind kits. Nanobind kits deliver ultra-clean, high-quality DNA that is ready for HiFi sequencing. That means no messy organic extraction. No challenges with phenol-chloroform extraction, like phase inversion, contamination, and degradation. And little to no DNA shearing.
How do Nanobind DNA extraction kits work?
The Nanobind DNA extraction protocol follows four basic steps:
- First, samples are lysed, using a specific buffer optimized for each sample type.
- DNA binds to Nanobind disks, which shield the DNA from damage during the extraction process.
- DNA is washed three times with ethanol-based buffers, removing contaminants.
- Finally, DNA is released from the Nanobind disk during elution without fragmentation, resulting in HMW DNA.
Top 8 DNA extraction tips for a better sequencing run
Here are a few best practices to keep in mind when you’re extracting DNA for HiFi sequencing.
1. Choose wisely
It may seem obvious, but if you have options when it comes to sample types, start by choosing the best one for the job. Blood is often the easiest sample type to work with for DNA studies. The volume of the sample required for DNA extraction depends on the species:
Sample volume required for DNA extraction
| Sample type | Whole blood volume for extraction | DNA source |
|---|---|---|
| Mammalian | 200 μL | White blood cells |
| Non-mammalian (e.g., birds, fish, reptiles) | 2.5-20 μL | Nucleated red blood cells |
2. Respect the input
Excessive input quantity can overwhelm the system chemistry, resulting in a less-than-optimal enzymatic reaction. This, in turn, lowers the quality of your resulting DNA.
3. Vortexing is your friend
For a more efficient lysis reaction, vortexing can help make sure your sample is well-mixed during the cell resuspension and lysis steps. This will improve DNA purity.
4. Keep it cold
Make sure to keep samples frozen when shipping and storing them before DNA extraction. Only saliva samples collected in Oragene™ devices (DNA Genotek) are stable at room temperature. For more information, read the technical note on saliva sample performance using Oragene devices and Nanobind kits.
5. Don’t let extraction “bug” you
The chitin found in exoskeletons can make it tricky to extract DNA from insect samples. To make this process easier, Nanobind extraction kits require only 30 mg of body mass (whether it’s the whole insect or just a body segment). For details about extracting HMW DNA from insect samples, see our technical note.
6. Wash it away
Size selection is key for long-read sequencing. It’s the first step of HiFi library preparation for whole-genome sequencing projects. Our Short Read Eliminator (SRE) kit will remove DNA fragments below 10 Kb. The SRE workflow uses simple size selective precipitation to pull HMW DNA out of solution by centrifugation. Low-molecular-weight (LMW) DNA is then washed away.
7. Garbage in, garbage out
Ultimately, higher DNA quality means better HiFi sequencing data. HMW DNA is essential for sequencing native DNA using HiFi reads. You’ll need > 2 µg of HMW DNA for whole-genome sequencing on the Vega system and > 500 ng HMW DNA on the Revio system.
8. Work smarter, not harder
If you need to extract a large number of blood, cell culture, or bacterial samples, the Nanobind CBB HT kit is compatible with automation.
Nanobind DNA extraction kits for any sequencing experiment
Now you’re ready to start DNA extraction from your sample for HiFi sequencing! Whether you’re focused on human DNA, animals, insects, plants, cell biology, or microbes, there’s a Nanobind kit to fit your study.
Nanobind PanDNA kit (PN 103-260-000)
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| Reactions | Automation options | Guide + overview | Protocol list |
| 24 | Manual | Nanobind PanDNA kit guide & overview | Nanobind PanDNA kit protocols list |
Nanobind CBB kit (PN 102-301-900)
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| Reactions | Automation options | Guide + overview | Protocol list |
| 24 | Manual | Nanobind CBB kit guide & overview | Nanobind CBB kit protocols list |
Nanobind CBB kit (PN 102-762-700)
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| Reactions | Automation options | Guide + overview | Protocol list |
| 96 |
KingFisher Apex System (Thermo Fisher) |
Nanobind HT kits guide & overview | Nanobind HT CBB kit protocols list |
Learn more about Nanobind kits and start your study on the right foot—with high-quality, high-molecular-weight DNA.
Complete workflows for long-read sequencing from PacBio
Extracting HMW DNA using Nanobind kits is just the beginning. PacBio has designed end-to-end workflows that scale with your study. From DNA extraction through sequencing, experts are available to support you with solutions for virtually any application, project, or research question.
See the workflow or connect with a PacBio scientist.