Pre-extraction sample processing for CALIBER project

Source steel beads (ball bearings) for tissue grinding (Tungsten beads are not usually necessary). We use hardened carbon steel or stainless steel bearings from simplybearings.co.uk. This protocol requires two beads per sample tube.

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.

Prepare 5 ml screwcap collection tubes (e.g. Starlab #E1450-1100) containing three 3 mm hardened steel beads in batches of 96 tubes.

Pick out a frozen plant sample bag from the freezer and note the sample name.

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.

Store at -20 ^oC until transported to SASA for qPCR analysis.

Write the sample name on a manilla sample drying envelope and find the corresponding dried sample label.

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.

The archive sample is ready for long term storage at room temperature.

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.

Store at -20 ^oC until ready to proceed with DNA extraction.

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.

Proceed with the next sample.