Aug 14, 2020
Manual DNA Extraction using Qiagen DNeasy Blood and Tissue Kit
- Julie Haendiges1,
- Ruth Timme1,
- George Kastanis1,
- Maria Balkey1
- 1US Food and Drug Administration
- GenomeTrakrTech. support email: genomeTrakr@fda.hhs.gov
Protocol Citation: Julie Haendiges, Ruth Timme, George Kastanis, Maria Balkey 2020. Manual DNA Extraction using Qiagen DNeasy Blood and Tissue Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.bi4dkgs6
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: July 28, 2020
Last Modified: November 10, 2021
Protocol Integer ID: 39781
Keywords: DNA Extraction, Qiagen Blood and Tissue Kit, GenomeTrakr, Whole Genome Sequencing,
Disclaimer
Please note that this protocol is public domain, which supersedes the CC-BY license default used by protocols.io.
Abstract
This SOP outlines the procedure for manual extraction of genomic DNA from live bacterial cells using the Qiagen Qiagen DNeasy Blood and Tissue Kit followed by quality control of purified extracts using the Qubit for purposes of whole genome sequencing.
This SOP is applicable to all GenomeTrakr laboratories with the intent of obtaining high quality, purified genomic DNA from gram-negative or gram-positive bacterial cells for use in subsequent sequencing protocols.
Complete in order:
1. DNA Extraction (included protocol or Automated DNA Extraction using the Qiacube)
- Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing
2. DNA Quantitation
- Quantitation of extracted DNA using the Qubit Flourometer
3. Library Preparation for WGS
- Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex)
4. Sequencing using Illumina MiSeq
5. Data Quality Checks and NCBI Submission
Guidelines
The Qiagen DNeasy Blood and Tissue Kit contains:
- AL Buffer
- ATL Buffer
- Proteinase K
- AW1 Buffer (Must be diluted prior to use, see "Before Start" section)
- AW2 Buffer ( Must be diluted prior to use, see "Before Start" section)
- Collection Tubes
- Spin Tubes
DNA Extractions used in WGS library preparation and sequencing must not contain EDTA. This chemical inhibits critical processes during library prep (ex. Tagmentation) and is found in the elution buffer contained in this kit. For this reason, Buffer EB should be ordered separately and used for final elution.
Materials
MATERIALS
Buffer EBQiagenCatalog #19086
DNeasy Blood & Tissue Kit, QIAGENCatalog #Cat No./ID: 69504
Proteinase K (2 ml)QiagenCatalog #19131
DNA LoBind TubesEppendorfCatalog ##022431021
1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
200 Proof Ethanol pureSigma AldrichCatalog #E7023
Lysostaphin - BioultraSigma – AldrichCatalog #L4402
Collection TubesQiagenCatalog #19201
Lysozyme from chicken egg white BioUltra lyophilized powderSigma AldrichCatalog #L4919
RNase AQiagenCatalog #19101
Triton X-100Fisher ScientificCatalog #MTX15681
0.5 M EDTAFisher ScientificCatalog #50-983-251
Equipment:
- Eppendorf 5430 Microcentrifuge (Eppendorf Catalog# 022620557 or equivalent)
- Incubator with 2.0 ml tube block attachment (Eppendorf Thermomixer Catalog# 2231000574 or a heatblock equivalent)
- Vortex
- Pipettes and tips
Safety warnings
Biosafety Warning: Foodborne pathogens are capable of causing serious disease. Always use a minimum of BSL2 practices, and use extreme caution when transferring and handling strains of this type. Wear appropriate PPE when handling infectious organisms. Work in a BSC when handling organisms with a low infectious dose. Disinfect or dispose of all plastic ware and glassware that are exposed to the bacterial cultures.
Chemical Safety Warning: Take the required precautions, and wear appropriate PPE when handling potentially hazardous chemicals. Ensure that chemicals, spent containers, and unused contents are disposed of in accordance with governmental safety standards.
RNase A has a GHS Category 1 classification as a skin and respiratory sensitization reagent. See the SDS for more information.
Qiagen DNeasy Kit Components: See Qiagen’s SDS for additional information.
AL Buffer: GHS Category 2 for skin & eye irritant. GHS Category 1 for skin sensitization. Contains a chaotropic salt and is not compatible with agents containing bleach.
AW1 Buffer: GHS Category 4 for oral & inhalation acute toxicity. GHS Category 2 for skin & eye irritation. Contains a chaotropic salt and is not compatible with agents containing bleach.
Proteinase K: GHS Category 1 for respiratory sensitization.
Before start
1. Bacterial Isolate Preparation: Sequencing should be preformed on pure isolated colonies only. Inoculate TSB with a single colony and incubate at 37oC for 16-24 hours.
2. Preparation of AW1 and AW2: These buffers need to be diluted with 100% Ethanol prior to use for the first time. Add the appropriate volumes as indicated on the bottle and write the date on the bottle.
3. Preheat incubator or heatblock to 56oC
Collect Cells
Collect Cells
Removed the inoculated TSB samples from the incubator.
Resuspend cells in the bacterial culture by pulse vortexing 3-5 times on low setting or by pipetting.
Transfer 1.0 mL bacterial culture into a 2.0 ml sterile Eppendorf tube.
Pellet cells by centrifuging at 7500 rpm for 00:05:00 minutes.
Discard supernatant into a 50 ml conical tube with 10% commercial bleach marked “biohazard waste".
Select the appropriate lysing conditions based on the type of bacteria:
Gram-Positive Bacteria26 steps
Prepare enzymatic lysis buffer (ELB) containing 20 mM Tris-HCl, 2 mM EDTA, and 1.2% Triton. A 250ml stock will be prepared. This stock buffer will last for 1 year at room temperature.
Mix 5 mL of 1 M Tris-HCl, 1 mL of 0.5 M EDTA, 3 mL of Triton X-100 and enough molecular-grade water to bring the volume up to 250 mL . Mix gently to incorporate the Triton.
Add 20 mg/ml of lysozyme to appropriate amount of lysis buffer within 24 hours of use.
Note: for Staphyloccocus spp., add 1 mg of lysostaphin for every 2 ml of enzymatic lysis buffer, regardless of ELB preparation method.
Add 180 µL of enzymatic lysis buffer (ELB) + Enzyme to sample tube.
Resuspend bacterial cell pellet by vortexing.
Incubate at 56 °C for 00:30:00 .
Add 4 µL of RNase A. Mix by vortexing and incubate at room temperature for 00:05:00
Add 25 µL of Proteinase K. Add 200 µL of Buffer AL. Vortex on high setting for 5 seconds to mix.
Incubate at 56 °C for at least 00:30:00
Add 200 µL of 100% Ethanol and mix by vortexing.
DNA Purification
DNA Purification
Label lid of a DNeasy spin column with a unique, recorded identifier for each sample.
Discard the collection tube and its contents.
Using a P1000 pipette, transfer full volume of pre-treated bacterial cells to the corresponding spin column.
Centrifuge at 8000 rpm (> 6000 x g) for 00:01:00
Place DNeasy spin column into a new 2.0 ml collection tube.
Add 500 µL of Buffer AW1.
Centrifuge at 8000 rpm (> 6000 x g) for 00:01:00
Discard the collection tube and its contents.
Place DNeasy spin column into a new 2.0 ml collection tube.
Add 500 µL of Buffer AW2.
Centrifuge at 13000 rpm (>20000 x g) for 00:03:00 .
Discard the collection tube and its contents.
Examine the spin column to ensure that no liquid remains on the spin column.
If there is liquid present, go to step #23 until it is clean.
Place spin column in a new, sterile 1.5 ml elution tube labeled with a unique, recorded identifier and/or FDA accession (CFSANxxxxxx) number.
Add 100 µL of Buffer EB to the spin column.
Optional: Incubate Buffer EB at 37°C to improve elution.
Incubate at room temperature for 00:05:00 .
Centrifuge at 8000 rpm (>6000 x g) for 00:01:00
DO NOT discard eluate, it is the purified DNA. DNA can be stored at 4 °C for short-term storage and -20 °C or -80 °C for long-term storage.