DNA shearing
Subsequently, DNA extracts need to be sheared to desired molecule sizes for optimal short-read sequencing. Short-read DNA sequencing platforms, such as Illumina sequencing, require DNA fragments to be inserted into sequencing adapters. These adapters are limited in size, typically accommodating fragments within a specific length range (e.g., 400 to 800 base pairs). Shearing the DNA to the desired fragment size ensures that the resulting library inserts are within the acceptable range for adapter ligation and subsequent sequencing. DNA shearing can be achieved using both physical methods, such as ultrasonication, and enzymatic digestion. Ultrasonication involves the use of high-frequency sound waves to break DNA molecules into smaller fragments. Enzymatic digestion involves the use of enzymes, such as restriction endonucleases or other DNA-cleaving enzymes, to break DNA molecules into smaller fragments. Each method has its advantages and considerations, and the choice between them depends on factors such as the desired fragment size range, sample type, and available equipment.
Instruments, plasticware and reagents
Instruments
- Covaris LE220 platform
- SAFE® Screw Cap De-/Capper (LVL technogies)
- SAFE® 2D/1D Code Reader (LVL technogies)
Plasticware
| Covaris 96-well plate |
Covaris |
PN 520272 |
| Plate-adhesive aluminium foil |
LVL |
AF100Plus |
Stock reagents
| Deionized ultrapure water (ddH2O) |
Bionordika |
BN-51100 |
4 ºC |
Protocol
- Calculate the amount of water and DNA extract volumes are required for each sample to obtain 200 ng of input DNA in 24 µl.
- Add the required volume of water to each well of the Covaris plate.
- Add the required volume of DNA extract to each well of the Covaris plate.
- Seal the plate with a self-adhesive aluminium foil.
- Quick-spin the plate to ensure the samples are at the bottom and all air bubbles are removed.
- Run DNA shearing using Covaris aiming for 450 nt-long DNA sequences.