We’ve developed a robust, cross-linked, chromatin shearing protocol for yeast samples, to be used prior to immunoprecipitation. Our protocol makes use of the Covaris E210, a Focused-ultrasonicator that is high throughput and decreases the probability of contamination relative to the Branson Digital Sonifier.
The M220 Focused-ultrasonicator uses the Adaptive Focused Acoustic (AFA) process to apply hydrodynamic shearing forces to the DNA to randomly fragment it. It is conducted under isothermal conditions ensuring both unbiased fragmentation and high recovery of double-stranded DNA.
This Application Note describes an Adaptive Focused Acoustics (AFA) procedure where three of the above procedures are combined into one step (1) the microbial cells are released from a solid matrix to which they are adhered, (2) the microbial cells are lysed, and (3) the DNA released from the cells is sheared to a desired fragment size with high precision and reproducibility.
Optimizing the sample preparation and chromatin shearing provides more chromatin available for the immunoprecipitation, preserves protein, epitopes, protein-DNA, interactions, and DNA quality.
During the development of DNA shearing protocols for Covaris Focused-ultrasonicators, the Agilent DNA 12000 kit was used to evaluate the average base pair (peak) size because of the large fragment size range this kit covers, encompassing the 130 bp to 5 kb range achievable with a Covaris system.
This Application Note describes the use of Covaris Adaptive Focused Acoustics (AFA) technology to extract DNA from Formalin-fixed, paraffin-embedded (FFPE) tissues.
Covaris has developed a novel method of active paraffin removal from Formalin-Fixed, Paraffin Embedded (FFPE) tissues utilizing Adaptive Focused Acoustics (AFA) technology. When an AFA treatment is applied to a FFPE sample, the paraffin is separated from the tissue sample by emulsification. This is an active mechanical process eliminating the use of hazardous organic solvents, or oils. It results in a robust and easily automatable workflow, which generates consistent DNA yield sample to sample, and improves the quality of the extracted nucleic acids; two parameters essential for clinical research and diagnostics.
Using yeast as a model organism, several commercially available native protein extraction reagents were tested for total protein yield and preservation of enzymatic activity using either a “passive extraction” method or an AFA-based “active extraction” method.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.