Jun 27, 2026
Column-Based DNA Extraction from Human Blood Clot Using Glass Bead Homogenization for Detection of Trypanosoma cruzi
- Ana Beatriz Barrero-García1,
- Luciana Basma2,
- Brandon N Mercado-Saavedra2
- 1Carrera de Ingeniería en Biotecnología, Universidad Católica Boliviana San Pablo;
- 2Instituto de Investigación de Medicina, Universidad Católica Boliviana San Pablo
Protocol Citation: Ana Beatriz Barrero-García, Luciana Basma, Brandon N Mercado-Saavedra 2026. Column-Based DNA Extraction from Human Blood Clot Using Glass Bead Homogenization for Detection of Trypanosoma cruzi. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v99jy4v3e/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: June 24, 2026
Last Modified: June 27, 2026
Protocol Integer ID: 319794
Keywords: Trypanosoma cruzi, DNA extraction, PCR, Blood clot, trypanosoma cruzi blood clot sample, dna extraction from human blood clot, chagas disease research, dna extraction, adapted dna extraction, based dna extraction, trypanosoma cruzi, human blood clot, recovery of dna, molecular detection, rna kit cwbio, pcr detection, suitable for downstream molecular analysis, challenging matrix for molecular detection, glass bead homogenization for detection, pcr detection in the context, derived pcr inhibitor, pcr inhibitor, downstream molecular analysis, dna
Funders Acknowledgements:
ANIDI FRICA
Grant ID: IIM-P6
Infectious Diseases Training program in Bolivia: South-South-North Training with Peru and JHU
Grant ID: 2D43TW010074-11
Abstract
Blood clot samples represent a challenging matrix for molecular detection of Trypanosoma cruzi due to their compact structure, high cellular content, and potential presence of blood-derived PCR inhibitors. This protocol describes an adapted DNA extraction workflow using the DNA/RNA Kit CWBIO (CWX101S), incorporating a mechanical clot disruption step prior to silica column-based purification. The method is designed to enhance sample homogenization and support the recovery of DNA suitable for downstream molecular analyses, particularly PCR detection in the context of Chagas disease research and diagnosis.
Protocol materials
Isopropyl alcohol
Lysis BufferJiangsu CowinCatalog #CWX101S
Proteinase KThermo Fisher ScientificCatalog #AM2548
Washing buffer 2Jiangsu CowinCatalog #CWX101S
Washing buffer 1Jiangsu CowinCatalog #CWX101S
Phosphate Buffered SalineMilliporeSigmaCatalog #P4417-100TAB
Bead-002-1K0LBX InstrumentsCatalog #21287
Safety warnings
In step 3, do not use a glass tube for vortexing the glass beads.
Before start
Preparation of Washing Buffers
- Prepare Washing Buffer 1 by adding 15 mL of isopropanol to the bottle labeled Washing Buffer 1.
- Prepare Washing Buffer 2 by adding 22.5 mL of absolute ethanol to the bottle labeled Washing Buffer 2.
- Close both bottles properly and mix gently by inversion 3-5 times until the solutions are completely homogenized.
- Verify that both bottles are correctly labeled and ready to use during the extraction procedure.
Sample preparation
5m
Obtain a Blood sample and allow it to coagulate.
Remove and discart the supernatant.
Add 20-25 Bead-002-1K0LBX InstrumentsCatalog #21287 and mix in a vortex for 00:05:00 to partially disrupt the clot.
5m
Sample Lysis
10m 15s
Take 100 µL of the previously disrupted clot and transfer it to a nuclease-free 1.5 mL Eppendorf tube.
Add 100 µL of 1X Phosphate Buffered SalineMilliporeSigmaCatalog #P4417-100TAB to bring the sample to a final volume of 200 µL , then vortex for 00:00:05 to homogenize.
5s
Add 200 µL of the Lysis BufferJiangsu CowinCatalog #CWX101S to the Eppendorf tube and vortex for00:00:05
5s
Add 20 µL of Proteinase KThermo Fisher ScientificCatalog #AM2548 to the tube and vortex again for 00:00:05
5s
Incubate the samples in a thermoblock at 65 °C for 00:10:00 to promote cell lysis and protein digestion.
10m
Precipitacion and transfer to the Extraction Colum
1m 10s
After incubation, add 300 µL of Isopropyl alcohol to each tube and vortex for 00:00:10
10s
Place a Silica Adsorption Column into its corresponding Collection tube.
Transfer the entire solution to the corresponding Adsorption Column.
Centrifuge for00:01:00 at 13400 x g
1m
Discard the flow-through from the Collection tube.
Colum Washing
6m
Mix the Washing buffer 1Jiangsu CowinCatalog #CWX101S bottle thoroughly by gentle inversion before use.
Add500 µL of Washing buffer 1Jiangsu CowinCatalog #CWX101S to each colum and centrifuge for 00:01:00 at 13400 x g
1m
Discard the flow-through from the Collection tube
Mix the Washing buffer 2Jiangsu CowinCatalog #CWX101S thoroughly by gentle inversion before use.
Add 500 µL of Washing buffer 2Jiangsu CowinCatalog #CWX101S to the Adsorption Colum and centrifuge at 13400 x g for 00:01:00
1m
Discart the flow-through from the Collection tube.
Place the Adsorption Column into the collection tube and centrifuge at 13400 x g for 00:02:00 to remove residual wash buffer.
2m
Elution
6m
Transfer the Adsorption Column to a clean elution 1.5 ml Eppendorf tube. Open the cap of the Adsorption Colum and allow it to air-dry at room temperature for 00:02:00
2m
Add 50 µL of RNase-Free Water directly to the middle of Adsorption Column and let stand at room temperature for00:02:00
2m
Centrifuge at 13400 x g for 00:02:00 to allow the elution of the nucleic acids.
2m
Proteinase K inactivation
10m
Incubate the eluted DNA at 90 °C for 00:10:00 to inactivate residual Proteinase KThermo Fisher ScientificCatalog #AM2548
10m
Store the extracted DNA at -80 °C to prevent degradation or use immediately for downstream molecular analyses.
Acknowledgements
The authors would like to acknowledge Franco Soto Mercado and Nicole Serrano from Carrera de Ingeniería en Biotecnología at Universidad Católica Boliviana San Pablo for their assistance in the development and implementation of this protocol. We also thank Mirian Cruz Zambana and Ana María Montaño from the Centro Nacional de Enfermedades Tropicales (CENETROP) for their valuable technical support and collaboration.