What is high molecular weight (HMW) DNA?
We define high molecular weight (HMW) as big DNA in the 50 - 300 kb size range. This is the perfect size for standard long-read sequencing on either PacBio or ONT. We usually lightly shear the DNA before library prep.
Some sample types may be slightly bigger or slightly smaller, but all Nanobind kits are designed to give consistent HMW DNA in this size range. They usually do not generate much small DNA less than 25 - 50 kb in length.
What is ultra high molecular weight (UHMW) DNA?
We define ultra high molecular weight (UHMW) as really big DNA in the 50 kb - 1+ Mb size range. This DNA is good for DNA mapping and for generating ultra-long megabase length reads or ultra high read length N50 (>150 kb).
The DNA size is similar to gel plug extracted DNA but can be obtained in only 1 - 2 hours. We currently have UHMW processes for cultured cells, bacteria, and human blood.
What has Nanobind HMW and UHMW DNA been used for?
Nanobind extracted HMW and UHMW DNA has been used for a variety of applications on PacBio, Oxford Nanopore, 10X, and Bionano Genomics platforms. It should be good for most applications that require high quality, high purity DNA.
It also works well for TaqMan qPCR.
What sample types can the Nanobind Big DNA Kits be used with?
The supported sample types for the Nanobind CBB Big DNA Kit include cultured cells, cultured bacteria, and human whole blood.
Our reference sample set, which is validated by long-read sequencing, comprises GM12878 cells,
listeria monocytogenes, and human whole blood (K2 EDTA, sodium citrate, heparin, and ACD stabilized).
Unsupported samples that this kit may also be used for, with appropriate modifications, include yeast and complex microbial samples.
The Nanobind Plant Nuclei Big DNA Kit allows isolation of high MW DNA from plant nuclei. This is an alpha kit that has only been tested with coastal redwood and sequoia needles.
The Nanobind Tissue Big DNA Kit has been used to extract an sequence a variety of animals including mammals, fish, crustaceans, birds, and insects.
We have additional kits in development for HMW DNA extraction from fungal, insect, and metagenomic sample types.
If you have an unsupported sample type, contact us. We may have an alpha kit you can test.
What is the maximum binding capacity of each Nanobind disk?
We don't know! We've never seen the limit. Due to Nanobind's unique format and binding mechanism, there may not be a limit.
We have bound as much as 10 mg of DNA to a single 5 mm Nanobind disk. We gave up after that. The image below shows what the Nanobind disk looks like with 50 ug, 1 mg, and 10 mg of DNA bound to it (from left to right).
If you can bind more DNA than this to a Nanobind disk, then let us know!
Are there any drawbacks to using HMW DNA?
HMW DNA has two handling complexities. First, it is viscous and gloopy. If your sample is gloopy and feels like snot, then you have big DNA on your hands (preferably in your tube). This means that you need to be extra careful and slow when pipetting to ensure that you don't shear your big DNA and that you transfer an accurate volume.
Second, high MW DNA can be heterogeneous. This means that to get an accurate concentration you have to perform multiple measurements from different locations within the tube (
i.e. top, middle, & bottom). Resting the sample at room temperature for 24 hours will often allow the DNA to relax and improve homogeneity.
The bigger the DNA, the more these issues will come into play.
See our Big DNA Handling Tips and Tricks if you need more help.
Can Nanobind be used for anything else other than big DNA?
Yes! We are developing Nanobind kits for many other applications in DNA/RNA sample prep. These kits will offer significant advantages in yield, nucleic acid quality, and purity. Stay tuned!
Can Nanobind extraction be automated?
Yes! Most of our development has been done using Thermo's KingFisher instrument. Identical HMW DNA can be obtained by either manual extraction or automated extraction. Scripts are provided on the Nanobind Support page.
Automation should be possible using pipetting type instruments but we haven't tested this yet.
Can Nanobind be used for reaction cleanup/purification?
Yes! We have a kit in development that is in early alpha testing now. Compared with magnetic particle cleanup, Nanobind cleanup is ultra gentle capable of generating megabase length reads without fragmentation.
In fact, we used it to generate the l
ongest sequencing read in the world at 2.44 Mb and to generate read length N50s up to 200 kb!