Aug 12, 2025

Public workspaceDNA Isolation from EDTA-blood

DOI
https://dx.doi.org/10.17504/protocols.io.14egnr2q6l5d/v1
  • Ann-Kathrin Hauser1,
  • Claudia Schulte1
  • 1AG Gasser, Hertie Inst. for Clinical Brain Research, University of Tübingen.
  • Ann-Kathrin Hauser: Neurobiobank of The Hertie Inst. for Clinical Brain Research
  • Claudia Schulte: Neurobiobank of The Hertie Inst. for Clinical Brain Research
Julia Fitzgerald
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DOI: https://dx.doi.org/10.17504/protocols.io.14egnr2q6l5d/v1
Protocol Citation: Ann-Kathrin Hauser, Claudia Schulte 2025. DNA Isolation from EDTA-blood. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnr2q6l5d/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: August 12, 2025
Last Modified: August 12, 2025
Protocol Integer ID: 224527
Keywords: dna isolation from edta, dna isolation, edta vial, total dna for long term storage, erythrocyte, total dna, edta, blood whole blood, whole blood, blood
Abstract
Whole blood collected in EDTA vials (9-10mL) is processed to remove the erythrocytes before salting out the total DNA for long term storage at 4 deg C.
Guidelines
All research must adhere to the current, updated Helsinki declaration.
Prior ethical approval must be granted.
Informed consent must be given.
Materials
Erylysis-Buffer                                                                                        
NH4Cl (molecular weight 53.49)                             41.45g
KHCO3 (molecular weight 100.12)                           5g
EDTA-solution (250mM, pH 7.4)                              2000µl
H2Od                                                                         up to 5L                                                                             
Store at 4°C
SE-Buffer                                                                                                 
NaCl (molecular weight 58.44)                                2.19g
EDTA-solution (250mM, pH 8)                                 50mL
H2Od                                                                          up to 500mL                                                                     
Store at room temperature
SDS-solution (10%)                                                                                
Autoclave, store at room temperature
TE Buffer (10x)                                                                                        
TRIS  (molecular weight 121.1)                                6.057g
EDTA-solution (250mM. pH 7.4)                            20mL
Adjust pH to 7.4
H2Od                                                                        Up to 500ml                                        
Autoclave, and store at room temperature
Protocol materials
ReagentS-Monovette®, K3 EDTASarstedtCatalog #02.1066.001
ReagentFalcon tube 50 mlThermo Fisher ScientificCatalog #1443222
Troubleshooting
Safety warnings
This protocol involves the processing of donated blood samples from routine hospital visits.
Ethical approval must be given by an IACUC or equivalent ethics committee prior to blood draw and processing.
Consent must be given by donors.
Ethics statement
The ethics committee of the Medical Faculty of the University of Tübingen and the University Clinic Tübingen (199/2011BO1 and 90/2009).


Before start
Ethical approvals in place.
Consent given.
Safety standards met for personnel to work with human blood.
Safety standards in place for disposal of human blood.
Removing the erythrocytes
Transfer whole blood from Amount9 mL ReagentS-Monovette®, K3 EDTASarstedtCatalog #02.1066.001 vial into Amount50 mL FalconReagentFalcon tube 50 mlThermo Fisher ScientificCatalog #1443222 and fill with Erylysis buffer (see materials tab) up to Amount50 mL volume.

Store for 10 minutes at Temperature-20 °C

Centrifigation1700 rpm, 4°C Centrifuge 10 minutes (1700rpm or 600g)

Equipment
Centrifuge 5810 R
NAME
Refrigerated centrifuge
TYPE
Eppendorf
BRAND
5811000065
SKU
https://www.eppendorf.com/gb-en/eShop-Products/Centrifugation/Multipurpose-Centrifuges/Centrifuge-5810-5810R-p-PF-240994
LINK


· Discard supernatant (in special waste container)
Safety information
All blood product waste should be treated according to local guidelines.

Fill the Falcon tube again with Erylysis-Buffer up to Amount30 mL and shake until the cell pellet has dissolved.

Store for 10 minutes at Temperature-20 °C

Centrifuge 10 minutes at 4°C at 1700rpm (600g) in The Eppendorf 5810R centrifuge.
Discard supernatant (in sink) and put the Falcon tube containing the cell pellet upside down onto a tissue (to dry).
Disruption of the cell pellet
Add Amount5 mL SE-Buffer (see materials tab) and vortex vigorously until pellet has dissolved

Add Amount500 µL 10% SDS-Buffer and Amount5 µL Proteinase K (20mg/ml) and vortex again.

Store tube at Temperature37 °C overnight or at TemperatureRoom temperature for 3 days.

Salting out
Add 2.5ml NaCl (5M) and vortex until it foams.
Centrifuge 10 minutes at 4°C at 2700rpm in The Eppendorf 5810R
If pellet is sharply defined, transfer supernatant into a new Amount50 mL Falcon tube, that has been previously filled with Amount15 mL 100% Ethanol.
(If pellet is not sharply seen, then add Amount500 µL NaCL (5M), vortex again and centrifuge again).

Rotate tube gently overhead to see DNA precipitating.
Fish the DNA to the side and discard the Ethanol.
Add Amount15 mL 70% Ethanol and gently shake.

Fish the DNA to the side and discard the Ethanol.
Add Amount15 mL 70%Ethanol and gently shake.

Transfer DNA to Cryotube (with pipette tip) and let it dry at room temperature.
Add Amount400 µL TE-Buffer (1X, see Materials tab) and shake at room temperature for at least 30 minutes.

Storage
Measure DNA
Store at Temperature4 °C