License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 06, 2022
Last Modified: May 06, 2022
Protocol Integer ID: 62111
Funders Acknowledgement:
BBSRC
Grant ID: BB/T010851/1
Abstract
This is a document that outlines the steps needed to prepare field-collected samples during the CALIBER project for archiving and further processing.
Materials
Plastics
2ml screwcap tubes
5ml fliptop tubes
15ml screwcap tubes
Tools
Scalpel handle and disposible blades e.g. Swann Morton #23 blades
fine forceps
premade labels for plant grinding tubes
premade labels for plant drying envelopes
premade labels for insect storage tubes
Safety warnings
This procedure involves the use of scalpels and sharp entomological forceps, ensure you are familiar with the relevant risk assessment for dissection in your organisation.
Beads are usually shipped coated in manufacturing oil (especially the carbon steel beads). To remove this, place beads in a borosilicate glass beaker or Duran bottle with the pouring lip and lid removed then bake for at least 12 hours at 250 oC.
Figure 1: Depending on baking time, carbon steel beads will change colour, this is normal.
Prepare 5 ml screwcap collection tubes (e.g. Starlab #E1450-1100) containing three 3 mm hardened steel beads in batches of 96 tubes.
Figure 2: Plant grinding tubes ready for plant material.
Insect sample processing
Insect sample processing
Pick out a frozen plant sample bag from the freezer and note the sample name.
Figure 3: Typical sample bags with sample names highlighted.
Using clean forceps, pick off all insects and place in a screwcap tube of the appropriate size (2ml/5ml/15ml) then fill the tube with a solution of 95% Ethanol and 5% Glycerol.
Label the tube with the corresponding tube label.
Figure 4: Insect tube labels.
Figure 5: Labelled tube with insects ready for storage.
Store at -20 oC until transported to SASA for qPCR analysis.
Figure 6: Box of insect tubes for longer term storage.
Archive plant sample processing
Archive plant sample processing
Write the sample name on a manilla sample drying envelope and find the corresponding dried sample label.
Figure 7: plant drying envelope and dried sample labels that are used to seal the flap.
Into this envelope, place approximately 2-5 g of leaf and chopped stem material.
Seal with the dried sample label and place in an oven overnight at 35-40 oC with a tray of silica gel in the bottom.
Figure 8: Plant sample drying over silica in the oven.
The archive sample is ready for long term storage at room temperature.
DNA extraction plant sample processing
DNA extraction plant sample processing
Using a clean scalpel, slice and place ~ 50 mg of plant material into the pre-prepared collection tubes containing the hardened steel ball bearings (from step 3).
Label the tube with the corresponding tube label.
Figure 9: Plant grinding tube with label, sample and ball bearings ready for storage.
Store at -20 oC until ready to proceed with DNA extraction.
Cleanup and reset
Cleanup and reset
The remaining plant material and packaging can be disposed of in a clinical waste bin.
Wash forceps and scalpel in 1x chemgene.
Rinse forceps and scalpel in molecular grade water.
Rinse forceps in 100% Isopropanol and leave to dry.
Wipe down dissection area with blue roll and 1x chemgene.