NGI offers support and help with DNA extractions, in particular when it comes to long-read sequencing, and difficult samples.
The NGI will normally not extract DNA for users unless previously agreed upon, but we are happy to advise our user on how to best extract DNA depending on sample type and sequencing requirements. The quality of the DNA is especially important for the success of long-read and linked-read methods, such as Nanopore and 10X Chromium. To extract high molecular weight DNA it is recommended to use kits or methods developed for this specific purpose. At NGI we have tried several of the commercially available options:
- MagAttract HMW (Qiagen)
- Nanobind CBB Big DNA Kit (Cells, Bacteria, Blood) (Circulomics)
- Nanobind Tissue Big DNA Kit (Circulomics)
- Fire Monkey (Revolugen)
It is important to handle the extracted HMW DNA with care to avoid fragmentation. This is done e.g by avoiding vortexing and unnecessary pipetting steps, using wide bore tips when possible and pipetting slowly. If you need advice on sampling and HMW DNA extraction, don’t hesitate to contact us!
Sample requirements, such as sample type and input amount, depend on the protocol used. In general DNA extracted from blood or cell cultures will have higher quality than DNA extracted from tissue. Fresh frozen samples are preferred when using tissue, but samples stored in ethanol may be used. To assure the quality of the sample it is important to avoid defrosting it once it has been frozen.
What we do with your samples
Most of the extraction methods used at NGI comprise the same four steps: lyse, bind, wash and elute. When the sample has been lysed, the DNA will bind to the beads, disc or column, depending on the protocol used. The wash steps will remove contaminants and inhibitors, after which the DNA will be eluted.
After extraction the yield is determined using fluorometric measurements (Qubit) and the quality is assessed by capillary electrophoresis (Femto Pulse, Fragment Analyzer).
The yield and quality of the extracted DNA depend on the sample type as well as the quality of the sample, thus no guarantees can be given regarding results. Successful extractions will result in HMW DNA suitable for most downstream applications.