Sanger Tree of Life Sample Preparation: Triage and Dissection
Caroline Howard, Juan Pablo Narváez-Gómez, Haddijatou Mbye, Michelle Strickland, Clare Cornwell, Halyna Yatsenko, Jessie Jay, Maria Morra
Abstract
This protocol describes the process of sample preparation for the extraction of DNA and/or RNA from the wide variety of samples processed by the Tree of Life Core Laboratory as part of the Tree of Life Programme. It also describes the triage steps and recommended weight requirements for the different taxonomic groups covered by the Tree of Life programme. The output of this protocol is a sufficient amount of sample that can be directed towards any of the Sanger Tree of Life Sample Homogenisation protocols.
Before start
Prepare at least 2 rubber buckets with dry ice (use more depending on the number of samples and other requirements/ personal preference):1. Cover one dry ice bucket with aluminium foil and place a metal dissection plate on top.
- Use the second dry ice bucket to store the samples that are going to be prepared. Samples should be kept on dry ice to ensure they remain frozen.
- Clean and pre-chill materials needed for dissection (weigh boats, scalpel, tweezers, scissors, etc.) by placing them on dry ice.
- Set up analytical balance.
- Retrieve frozen samples.
Steps
Laboratory procedure
Select the first sample from the cold rack.
Inspect the sample and check the sample preservation method.
For snap frozen samples (‘standard samples’), proceed to step 3.
For samples that have not been snap frozen (‘non-standard samples’), please refer to step 7.
Decide how much sample is required for the desired downstream process (see Table 1 or the relevant Decision Making Tree in Appendices).
Weigh the sample. If taking only part of a sample, dissect the desired amount.
- Use an analytical scale to tare a new clean weighing boat.
- Use clean and pre-chilled dissection tools (scalpel, tweezers, scissors) and metal dissection block to cut the sample until the desired weight has been achieved.
- Place prepared sample into correct destination container and put back any remaining tissue into original container
- Clean the tools and dissection block with Azowipe tissue and bury the dissection tools in dry ice so that they are ready for the next sample.
If the total weight is 25 mg or less and it is from Metazoa (excluding sponges and corals), use the ‘Sanger Tree of Life Sample Homogenisation: PowerMash’ protocol.
If the total weight is greater than 25 mg for all taxonomic groups, use ‘Sanger Tree of Life Sample Homogenisation: Covaris cryoPREP® Automated Dry Pulverizer’ protocol.
The current method to prepare samples stored in preservative solutions is as follows:
- Place the tube containing the sample in wet ice to allow the liquid to defrost.
- Remove the sample from solution and transfer to a new tube. Dispose of the preservative solution in an appropriate waste container.
- Then proceed as if the sample was frozen tissue (see step 3). Keeping in mind the recorded weight will not be accurate, as the sample will have absorbed some of the preservative solution.
If the sample type is blood, defrost on wet ice and aliquot required amount for the desired downstream protocol. If the blood is stored in ethanol, the aliquot should be taken from underneath the layer of ethanol.
