Insect sample preparation for RE-Pore-C
Extraction Method
Insect sample preparation for RE-Pore-C
FOR RESEARCH USE ONLY
Contents
Introduction
Materials
Results
Introduction
This protocol describes the preparation of a sample of Drosophila melanogaster (D. melanogaster) to be processed using the restriction enzyme Pore-C (RE-Pore-C) protocol as an example of insect material. This protocol was developed using D. melanogaster white eye mutant variant W1118 at the adult stage.
Materials
- 100 mg of insect material
- 1X PBS pH 7.4
- Crushed ice
- Liquid nitrogen
- 15 or 50 ml centrifuge tubes
- Mortar and pestle
- –80°C freezer storage
Cryogrinding of insect material: 10 minutes hands-on-time
Note: Pre-cool the mortar and pestle at –80°C for at least 30 minutes. Fresh and frozen samples may be used.
Gather 100 mg of insect material. Note: If the sample of insect material is >1 cm^2, dissect the sample into smaller pieces before proceeding to the next step.
Place the chilled mortar and pestle on ice. Pour a small volume of liquid nitrogen into the mortar and add the sample of insect material to freeze until the liquid nitrogen has evaporated.
Carefully grind the frozen insect material into a fine powder, working quickly to minimise thawing. If the material starts to thaw, add another small volume of liquid nitrogen to the mortar.
Use a spatula to collect the insect powder into a chilled centrifuge tube on ice.
RE-Pore-C extraction
Transfer approximately 100 mg of cryo-ground tissue to a 50 ml centrifuge tube and resuspend in 1 ml chilled 1X PBS.
Bring the volume of the re-suspended cryo-ground tissue to 10 ml in chilled 1X PBS.
Proceed with the RE-Pore-C protocol using the re-suspended cryo-ground tissue powder as input.
Results
| Sample | DNA concentration, ng/μl | Total DNA mass, μg |
|---|---|---|
| D. melanogaster | 31.8 | 4.77 |
Table 1. The yield of non-size selected RE-Pore-C DNA extract using NlaIII restriction enzyme.
Figure 1. Agilent Bioanalyser DNA 12000 trace of non-size selected RE-Pore-C DNA extract.
Figure 2. The sequencing and Pore-C output for libraries assessed on PromethION. Libraries were generated as described using Pore-C extracts prepared with the NlaIII restriction enzyme. The read length distribution and output (Gbases) obtained from the libraries generated are shown in panels A and B, respectively. Panel C displays the Pore-C metrics obtained.