We demonstrate that intact RNA can be efficiently extracted from Mycobacteria smegmatis using the Covaris Adaptive Focused Acoustics® (AFA®) M220 or ME220 Focused-ultrasonicators. By applying precise control of AFA power conditions, RNA can be differentially extracted to favor intact subunits or shorter fragments. AFA technology can effectively replace bead-beating protocols for extraction of RNA. AFA extraction methods are more consistent, provide high quality RNA and are easy to perform.
Ranjan et. al., (1) described the process for mechanically shearing DNA to 300-600 bp fragments with a Covaris® S220 Focused-ultrasonicator. The shearing process was done on DNA previously
extracted and frozen. To achieve high level of reproducible shearing, the Covaris S220 Focused-ultrasonicator with SonoLab software controls critical parameters such as Peak Incident Power
(PIP), Duty Factor (DF), Cycles Per Burst (CPB), temperature and duration of treatment.
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.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.