Sanger Tree of Life Sample Homogenisation: PowerMash
Michael Quail, Caroline Howard, graeme oatley, Clare Cornwell, Amy Denton
Abstract
This protocol describes the homogenisation of tissue samples for DNA and/or RNA extraction intended for long read sequencing or RNA-Seq, using the Diagnocine PowerMasher II tissue disruptor. This process is highly effective for the disruption of tissue samples weighing less than 25 mg from all taxonomic groups covered by the Tree of Life Programme except for plants, fungi, protists, sponges and corals. Larger samples are not processed through this method due to difficulties in achieving homogenisation. Crustaceans and molluscs are particularly challenging samples. The output of this protocol is a sample that can be directed toward any of the Sanger Tree of Life DNA and RNA extraction protocols.
Before start
Prepare lysis buffer for the tissue’s intended downstream protocol: either DNA or RNA extraction.
Steps
Laboratory protocol
Transfer the sample into the sterile 1.5 mL BioMasher II tube and add the lysis buffer required for the downstream protocol.
Attach the sterile BioMasher pestle to the PowerMasher II tissue disruptor, avoiding contact with the end of the pestle that will contact the sample to prevent contamination.
Disrupt the sample in the lysis buffer within the BioMasher tube using the PowerMasher II tissue disruptor and sterile BioMasher pestle; this is done by placing the pestle into the BioMasher tube and against the sample, applying some gentle pressure so the sample remains between the pestle and the wall of the BioMasher tube, and then squeezing the trigger of the PowerMasher II tissue disruptor to activate it.
Continue for approximately 30 seconds to 1 minute (depending on the tissue type), until no large pieces of the sample remain, or the sample cannot be disrupted any further. Gently moving the pestle up and down can encourage sample movement within the lysis buffer and prevents the sample becoming stuck at the bottom of the tube, allowing for maximum disruption.
Remove the pestle from the BioMasher tube and disconnect it from the PowerMasher tissue disruptor II. If any tissue remains stuck to the pestle, gently try to remove this by wiping the pestle tip on the rim of the BioMasher tube. Dispose of pestle in biological waste.
Proceed with the powermashed sample to the next step of the tissue’s downstream protocol –
either DNA or RNA extraction.
