Jan 03, 2025

Public workspaceDNA extraction in Theridion evexum

DOI
dx.doi.org/10.17504/protocols.io.kxygxwp2kv8j/v1
  • Ruth Madrigal-Brenes1,
  • Gilbert Barrantes1,
  • Luis Sandoval1,
  • Eric J. Fuchs1
  • 1Universidad de Costa Rica
Eric J. Fuchs
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DOI: dx.doi.org/10.17504/protocols.io.kxygxwp2kv8j/v1
Protocol Citation: Ruth Madrigal-Brenes, Gilbert Barrantes, Luis Sandoval, Eric J. Fuchs 2025. DNA extraction in Theridion evexum. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwp2kv8j/v1
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: November 22, 2024
Last Modified: January 03, 2025
Protocol Integer ID: 112656
Keywords: microsatellites, tropical spiders, CTAB, Costa Rica
Funders Acknowledgements:
Vicerrectoría de Investigación
Grant ID: C3-116
Abstract
The number of species threatened by fragmentation and loss of natural habitats due to changes in land use and unplanned urban expansion are rapidly increasing. Despite the seriousness of the situation, information on the impact of isolation caused by fragmentation and urbanization on spider genetic diversity is scarce, owing mostly to a lack of appropriate molecular markers. The main objective of this study was to develop microsatellite (SSR) primers for the spider Theridion evexum using low-coverage next-generation sequencing and bioinformatic tools. To increase the yield of DNA extracted from small spiders like T. evexum, we also optimized a CTAB DNA extraction protocol. We sequenced eight individuals at 4X using paired-end sequencing on an Illumina Novaseq 6000. Reads were cleaned and processed using the MiMi python pipeline. MiMi produced a total of 3999 putative microsatellite primers. After filtering for polymorphic loci with an allelic richness greater than three and primers that were present in at least 5 of the 8 sequenced individuals, 34 final markers were identified. An in vivo test of 13 of these 34 markers showed that 10 loci were polymorphic with at least three detectable alleles, one locus was monomorphic, and two loci did not produce PCR products. These markers will allow a better assessment of the effects of fragmentation and isolation across populations of this spider species. Furthermore, developing markers using low-coverage NGS (next-generation sequencing) and bioinformatic methods provide a valuable approach for uncovering SSR markers at a reduced cost for other tropical species, thereby broadening the scope of molecular ecology research in the tropics.

Protocol materials
ReagentEDTA disodium dihydrateAbblisCatalog #AB1011793
Step 8
ReagentSodium ChlorideCatalog #PubChem CID: 5234
Step 8
ReagentProteinase K (20 mg/ml)
Step 9
ReagentIsopropanol
Step 17
ReagentLiquid nitrogen
Step 6
ReagentPolyvinylpyrrolidone (PVP-40)Bio Basic Inc.Catalog #PB0436.SIZE.250g
Step 8
ReagentCTAB
Step 8
ReagentChlorofom-octanol (24:1)
Step 13
ReagentTE Buffer
Step 28
Reagent70% Ethanol
Step 22
Sample preparation
Sample preparation
Remove the sample from the alcohol in which it was preserved and place it in a Petri dish under the stereoscope.
Remove the sample’s abdomen and discard it, leaving only the cephalothorax and legs.
Place the cephalothorax and legs (called from now on “the sample”) on a piece of paper towel to remove as much alcohol as possible from the sample.
Leave the sample at room temperature for 5 minutes to allow any remaining alcohol to evaporate.
DNA extraction
DNA extraction
3h 24m 5s
3h 24m 5s
Transfer the sample to a pre-chilled (with liquid nitrogen for 15 seconds) 1.5 mL microcentrifuge tube.
Add approximately Amount1 mL ReagentLiquid nitrogenContributed by users to freeze the sample completely.

Ground the sample into small pieces (< 1 mm) or a fine powder using a plastic pestle.
AddAmount500 µL CTAB buffer (Concentration2 % volume CTAB: Concentration100 millimolar (mM) Concentration1.4 Molarity (M) ReagentSodium ChlorideContributed by usersCatalog #PubChem CID: 5234 , Concentration20 millimolar (mM) ReagentEDTA disodium dihydrateAbblisCatalog #AB1011793 , Concentration2 Mass / % volume ReagentCTABContributed by users , Concentration2 % (v/v) ReagentPolyvinylpyrrolidone (PVP-40)Bio Basic Inc.Catalog #PB0436.SIZE.250g )

Add Amount20 µL of ReagentProteinase K (20 mg/ml)Contributed by users

IncubateDurationOvernight at Temperature55 °C with continuous shaking at 800 rpm.

1. Centrifuge samplesCentrifigation14000 rpm, Room temperature, 00:05:00

5m
Transfer Amount400 µL of the supernatant to a 2 mL tube.


Add Amount400 µL of ReagentChlorofom-octanol (24:1)

Mix by inverting the tubes forDuration00:02:00

2m
Centrifuge samples Centrifigation14000 rpm, Room temperature, 00:05:00

5m
Transfer 300 µL of the supernatant (without disturbing the organic phase) to a 1.5 mL tube.
Add Amount300 µL of ReagentIsopropanolContributed by users at Temperature-20 °C .

Mix by inverting the tubes for Duration00:02:00 .

2m
Place the samples on ice and refrigerate at Temperature-20 °C for Duration01:00:00 (or overnight).

1h
Centrifuge samples Centrifigation14000 rpm, Room temperature, 00:05:00
5m
Discard all the supernatant ensuring not to disturb the pellet (important: remove supernatant as much as you can).
Add Amount500 µL Reagent70% EthanolContributed by users .

Mix by vortex for Duration00:00:05 .

5s
Centrifuge samples Centrifigation14000 rpm, Room temperature, 00:05:00

5m
Discard all the ethanol by decantation ensuring not to disturb the pellet (important: remove alcohol as much as you can).
Repeat steps 22-25 at least once again.

Leave the tubes upside down on a napkin to remove excess ethanol for Duration02:00:00

2h
Once the pellet was completely dry, it was eluted in Amount50 µL of ReagentTE BufferContributed by users .