DNA isolation protocols per biobank
Auria Biobank - protocol for DNA isolation from whole blood samples
15.3.2022
Kit: Chemagic DNA Blood 400
Sample: Concentrated fresh whole blood drained to 10 ml EDTA tube
Elution volume: 150 μl
Elution buffer: Elution Buffer from the kit
Consumables: Basic plastic ware from the kit, 96 well skirted PCR plate (4titude) + heat seal (4titude) for DNA, RNase/DNase free & non pyrogenic tips with filters
Protocol: Purification Protocol for 400 µl of Human Blood using the Chemagic 360 (Perkin Elmer) with minor modifications
Sample batch: 96 samples
Quality controls: Concentration and purity (A280/A260 & A260/A230 ratio) measured with Tecan Spark microplate reader. IBBL proficiency test for DNA Extraction from Whole Blood every year since 2017.
Note: -
Biobank of Eastern Finland - protocol for DNA isolation from buffy coat samples
18.3.2022
Kit: Macherey-Nagel, NucleoSpin 96 Tissue Core Kit (4x96 preps) 740454.4
Sample: Buffy coat prepared from 10 ml of EDTA blood (centrifuged 10 min, 2200rcf, RT, divided into aliquots of 220 µl, mainly within 24 h of phlebotomy (up to app. 72 h), aliquots stored at -20°C or colder, thawed for subsequent DNA extraction)
Elution volume: 160 µl (2 elution rounds of 80 µl to one tube)
Elution buffer: BE elution Buffer from kit: 5mM Tris/HCL, pH 8.5
Consumables: Pipette tips, 60 ml Reagent Reservoirs, 120 ml Reagent containers, Wash plate
Protocol: NucleoSpin® 96 Tissue – vacuum processing
https://www.mn-net.com/media/pdf/0a/17/0b/Instruction-NucleoSpin-96-Tissue.pdf
The protocol is optimized for the automated processing on Hamilton Microlab STAR liquid handler at BBEF. Changes done to the manufacturer’s protocol:
Samples used: 200µl Buffy coat
Lysis buffer: B3 (ref: 740920) instead of T1
Lysis step by shaker 1800rpm/30min/70°C
Binding conditions: only Ethanol and shaker 1500rpm/2min/RT
Elution step twice (2min/200mbar) with preheated (+70°C) elution buffer BE (2x80µl)
Sample batch: 96 buffy coat samples.
Quality controls: DNA concentration and purity (A260/A280 and A260/A230 ratio) is measured with Denovix-DS-11 (or VictorNivo Microplate Reader, A260/A280)
Note: Until 04/2021 quality controls also included DNA concentration measurement with Qubit. Currently fluorescence measurements done occasionally if needed/asked by a customer.
FRC Blood Service Biobank - protocol for DNA isolation from blood samples
3.6.2022
Kit: QIAsymphony DSP DNA Midi Kit (937255)
Sample: 1.2 ml of whole blood from 4.0 ml EDTA tube (fresh or frozen at -20°C and thawed for DNA extraction)
Elution volume: 1000 μl or 400 µl
Elution buffer: Qiagen ATE buffer (10 mM Tris-Cl pH 8.3, 0.1 mM EDTA, 0.04% NaN3 (Sodium-azide)
Consumables: Standard QIAsymphony plastic ware including magnetic rod covers, sample preparation cartridges and pipette tips
Protocol: Biopankki1000 or B400
Sample batch: QIAsymphony isolates DNA in batches of 4x 24 extractions. In our standardized protocol, one isolation run consists of 96 donor samples.
Quality controls: DNA concentration and purity (A280/A260) is measured with Dropsense96 microplate reader
Note: QIAsymphony DNA Kits co-purify DNA and RNA
Central Finland Biobank - protocol for genomic DNA isolation from whole blood samples
16.3.2022
Kit: PerkinElmer chemagic DNA Blood 250 Kit H96 (CMG-746)
Sample: 200 µl of whole blood aliquoted from 9 ml of EDTA blood. Primary samples are collected in room temperature, stored at 4 °C and aliquoted within 72 hours of phlebotomy, after which the aliquots are stored long term at -70 °C or liquid nitrogen (vapor phase). Short term storage (2-3 weeks) in -20°C. Aliquots are thawed at 37 °C for 30 minutes prior to DNA extraction.
Elution volume: 100 µl
Elution buffer: PerkinElmer chemagen Elution Buffer 7 (10 mM Tris-HCl pH 8.0)
Consumables: Standard chemagic 360 plasticware, including magnetic rod covers as well as deep well plates for sample preparation and low well plates for magnetic beads. DNA delivered to THL in foil sealed Thermo Scientific PCR plates (AB0800, 96-well, low profile, skirted).
Protocol: chemagic DNA Blood 250 360 H96 prefilling VD190917
Sample batch: 96 samples per extraction run
Quality controls: DNA concentration and purity (A280/A260 and A260/A230 ratio) are measured with Thermo Scientific NanoDrop 2000c spectrophotometer.
Note: -
Finnish Clinical Biobank Tampere - protocol for DNA isolation from EDTA blood samples
28.3.2022
Kit: sbeadex® blood kit (LGC, Biosearch Technologies), adapted protocol to higher blood volume
Samples: 2-3 mL human EDTA blood, stored in barcoded screw cap tubes
Elution volume: 280 - 300 μL
Elution buffer: 10 mM Tris, pH 8.0
Consumables: Matrix 2D barcoded screw top tubes (cat. no. 3745) + caps (cat.no. 4477)
1 x normalized DNA aliquot: 330µL MTP, V-bottom, PP, (4titude, cat. no. 4ti-0117)
DNA normalization: on Hamilton STAR/STARlet liquid handling systems.
Normalization concentration: 50ng/µL Aliquots
Protocol: LGC protocol for DNA isolation from EDTA blood samples, stored at -80°C. DNA extraction from 2-3 mL blood with sbeadex blood kit on KingFisher 96/Flex (Finnish Biobank). Semi-automatic method: blood lysis and binding to the magnetic beads in 15mL tubes, 4 washing steps and elution of the DNA on KingFisher 96/Flex robot.
Sample batch: 96 blood samples per extraction
Quality controls: DNA concentration and purity (A280/A260 and A260/A230 ratio) is measured with Nanodrop 8000 Spectrophotometer (ThermoFisher). Agarose gel check and PCR of 2% of all extracted DNA samples
Note: Tampere Biobank has outsourced DNA isolation to LGC Technologies (LGC Genomics GmbH).
Helsinki Biobank - protocol for DNA isolation from buffy coat samples
8.3.2022
Kit: QIAsymphony DSP Midi kit
Sample: Buffy coat prepared from 10 ml of EDTA blood (centrifuged 10 min, 2000 g, +4°C, divided into aliquots of 500 μl and 300 μl within 24 h of phlebotomy, aliquots stored at -80°C or colder, thawed for subsequent DNA extraction)
Elution volume: 200 μl
Elution buffer: Until April 1, 2022: Qiagen ATE buffer (10 mM Tris-Cl pH 8.3, 0.1 mM EDTA, 0.04% NaN3 (Sodium-azide). Since April 1, 2022: Qiagen DNA elution buffer (10 mM Tris, pH 8.5).
Consumables: Standard QIAsymphony plastic ware including magnetic rod covers, sample preparation cartridges and pipette tips
Protocol: BC400 CR22484
Sample batch: QIAsymphony isolates DNA in batches of 24 extractions. In our standardized protocol, one batch consists of 23 patient samples and 1 negative control (water).
Quality controls: DNA concentration and purity (A280/A260 and A260/A230 ratio) is measured with TECAN SPARK multimode microplate reader
Note: QIAsymphony DNA Kits co-purify RNA and DNA. Prior to April 1, 2022, no RNase treatment was included. Since April 1, 2022, 10 μl RNAseA from a 100 mg/ml RNAseA stock solution was added to each buffy coat sample before loading samples into QIAsymphony.
Northern Finland Biobank Borealis - protocol for DNA isolation from whole blood samples
28.3.2022
Kit: QIAsymphony DSP Midi kit
Sample: Whole blood prepared from 10 ml of EDTA blood: an aliquot of 1700 µl transferred to a separate tube before centrifugation steps to generate plasma aliquots (centrifuged 10 min, 2000 g, +4°C). Alternatively (fall 2021 onwards) 3 ml EDTA tube is directly used in the extraction protocol. In both cases, the extraction is done using 1 ml of whole blood. Freezing time from phlebotomy varies due to long distances in the biobank's geographical area. Aliquots stored at -80°C or colder, thawed for subsequent DNA extraction.
Elution volume: 180 μl and 19.7.2017 onwards 235 µl
Elution buffer: Qiagen ATE buffer (10 mM Tris-Cl pH 8.3, 0.1 mM EDTA, 0.04% NaN3 (Sodium-azide)
Consumables: Standard QIAsymphony plastic ware including magnetic rod covers, sample preparation cartridges and pipette tips
Protocol: Blood 1000 DSP
Sample batch: QIAsymphony isolates DNA in batches of 24 extractions
Quality controls: DNA concentration and purity (A280/A260 and A260/A230 ratio) are measured with QIAxpert
Note: No RNase treatment was included
Terveystalo Biobank - protocol for DNA isolation from whole blood samples
15.3.2022
Kit: Chemagic DNA Blood 400
Sample: Concentrated fresh whole blood drained to 10 ml EDTA tube
Elution volume: 150 μl
Elution buffer: Elution Buffer from the kit
Consumables: Basic plastic ware from the kit, 96 well skirted PCR plate (4titude) + heat seal (4titude) for DNA, RNase/DNase free & non pyrogenic tips with filters
Protocol: Purification Protocol for 400 µl of Human Blood using the Chemagic 360 (Perkin Elmer) with minor modifications
Sample batch: 96 samples
Quality controls: Concentration and purity (A280/A260 & A260/A230 ratio) measured with Tecan Spark microplate reader. IBBL proficiency test for DNA Extraction from Whole Blood every year since 2017.
Note: Terveystalo Biobank has outsourced DNA isolation to Auria Biobank
THL Biobank - protocol for DNA extraction from blood/saliva samples
17.3.2022
Kit: Chemagen blood/saliva kit
Sample: Whole blood (4-9ml) extracted either fresh or frozen. Storage time from months to decades, storage temperature -20°C. Saliva collected in Oragene OG-500 collection tube, stored in RT.
Elution volume: 400-1000 μl
Elution buffer: 10mM Tris-EDTA, pH 8
Consumables: Standard Chemagen plastic ware including magnetic rod covers, sample preparation cartridges and pipette tips
Protocol: VD150310.che 5ml blood samples
VD150513.che 10ml blood samples
VD161026.che saliva samples
Sample batch: Chemagen360 and Janus-Chemagen PRO isolates DNA in batches of 12 or 24 extractions
Quality controls: DNA concentration and purity (A280/A260 and A260/A230 ratio) is measured with NanoDrop or Trinean DropSense. In normalization procedure DNA is quantified using PicoGreen.
Note: -
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