TITLE: Manual DNA Extraction using Qiagen DNeasy Blood and Tissue Kit
AUTHORS: Julie Haendiges, Ruth Timme, George Kastanis, Maria Balkey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><div class = "text-block">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.</div><div class = "text-block"><span style = "font-weight:bold;">Complete in order:</span></div><div class = "text-block"><span style = "font-weight:bold;">1. DNA Extraction (included protocol </span><span style = "font-weight:bold;font-style:italic;">or</span><span style = "font-weight:bold;"> Automated DNA Extraction using the Qiacube)</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">2. DNA Quantitation</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Quantitation of extracted DNA using the Qubit Flourometer </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">3. Library Preparation for WGS</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Library preparation using NexteraXT </span><span style = "font-style:italic;">or </span><span>Illumina DNA Prep (previously Nextera DNA Flex)</span></li></ul></div><div class = "text-block"><span style = "font-weight:bold;">2. DNA Quantitation</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Quantitation of extracted DNA using the Qubit Flourometer </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">3. Library Preparation for WGS</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Library preparation using NexteraXT </span><span style = "font-style:italic;">or </span><span>Illumina DNA Prep (previously Nextera DNA Flex)</span></li></ul></div><div class = "text-block"><span style = "font-weight:bold;">3. Library Preparation for WGS</span></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block"><span style = "font-weight:bold;">4. Sequencing using Illumina MiSeq</span></div><div class = "text-block"><span style = "font-weight:bold;">5. Data Quality Checks and NCBI Submission</span></div></div>

[STEPS]
?. [Collect Cells]
Resuspend cells in the bacterial culture by pulse vortexing 3-5 times on low setting or by pipetting.
?. [Collect Cells]
Transfer  bacterial culture into a 2.0 ml sterile Eppendorf tube.
1 mL
?. [Collect Cells]
Pellet cells by centrifuging at  for  minutes.
Centrifuge: 7500 33
?. [Collect Cells]
Discard supernatant into a 50 ml conical tube with 10% commercial bleach marked “biohazard waste".
?. [Lysing Bacterial Cells]
Select the appropriate lysing conditions based on the type of bacteria:
?. [Lysing Bacterial Cells]
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  of 1 M Tris-HCl,  of 0.5 M EDTA,  of Triton X-100 and enough molecular-grade water to bring the volume up to . Mix gently to incorporate the Triton.
5 mL
1 mL
3 mL
250 mL
?. [Lysing Bacterial Cells]
Add l of Buffer ATL. (Check for precipitate in ATL, if precipitate is present, warm to 56oC until it is fully dissolved)
180 µl
?. [DNA Purification]
Label lid of a DNeasy spin column with a unique, recorded identifier for each sample.
?. [DNA Purification]
Using a P1000 pipette, transfer full volume of pre-treated bacterial cells to the corresponding spin column.
?. [DNA Purification]
Centrifuge at  (> 6000 x g) for
Centrifuge: 8000 33
?. [DNA Purification]
Discard the collection tube and its contents.
?. [DNA Purification]
Place DNeasy spin column into a new 2.0 ml collection tube.
?. [DNA Purification]
Add  of Buffer AW1.
500 µl
?. [DNA Purification]
Centrifuge at  (> 6000 x g) for
Centrifuge: 8000 33
?. [DNA Purification]
Discard the collection tube and its contents.
?. [DNA Purification]
Place DNeasy spin column into a new 2.0 ml collection tube.
?. [DNA Purification]
Add  of Buffer AW2.
500 µl
?. [DNA Purification]
Centrifuge at  (>20000 x g) for .
Centrifuge: 13000 33
?. [DNA Purification]
Discard the collection tube and its contents.
?. [DNA Purification]
Examine the spin column to ensure that no liquid remains on the spin column. If there is liquid present,   until it is clean.
?. [DNA Purification]
Place spin column in a new, sterile 1.5 ml elution tube labeled with a unique, recorded identifier and/or FDA accession (CFSANxxxxxx) number.
?. [DNA Purification]
Add  of Buffer EB to the spin column.Optional: Incubate Buffer EB at 37°C to improve elution.
100 µl
?. [DNA Purification]
Incubate at room temperature for .
?. [DNA Purification]
Centrifuge at  (>6000 x g) for
Centrifuge: 8000 33
?. [DNA Purification]
DO NOT discard eluate, it is the purified DNA. DNA can be stored at  for short-term storage and  or  for long-term storage.
4 °C
-20 °C
-80 °C
?. [DNA Purification]
Label lid of a DNeasy spin column with a unique, recorded identifier for each sample.
?. [DNA Purification]
Using a P1000 pipette, transfer full volume of pre-treated bacterial cells to the corresponding spin column.
?. [DNA Purification]
Centrifuge at  (> 6000 x g) for
Centrifuge: 8000 33
?. [DNA Purification]
Discard the collection tube and its contents.
?. [DNA Purification]
Place DNeasy spin column into a new 2.0 ml collection tube.
?. [DNA Purification]
Add  of Buffer AW1.
500 µl
?. [DNA Purification]
Centrifuge at  (> 6000 x g) for
Centrifuge: 8000 33
?. [DNA Purification]
Discard the collection tube and its contents.
?. [DNA Purification]
Place DNeasy spin column into a new 2.0 ml collection tube.
?. [DNA Purification]
Add  of Buffer AW2.
500 µl
?. [DNA Purification]
Centrifuge at  (>20000 x g) for .
Centrifuge: 13000 33
?. [DNA Purification]
Discard the collection tube and its contents.
?. [DNA Purification]
Examine the spin column to ensure that no liquid remains on the spin column. If there is liquid present,   until it is clean.
?. [DNA Purification]
Place spin column in a new, sterile 1.5 ml elution tube labeled with a unique, recorded identifier and/or FDA accession (CFSANxxxxxx) number.
?. [DNA Purification]
Add  of Buffer EB to the spin column.Optional: Incubate Buffer EB at 37°C to improve elution.
100 µl
?. [DNA Purification]
Incubate at room temperature for .
?. [DNA Purification]
Centrifuge at  (>6000 x g) for
Centrifuge: 8000 33
?. [DNA Purification]
DO NOT discard eluate, it is the purified DNA. DNA can be stored at  for short-term storage and  or  for long-term storage.
4 °C
-20 °C
-80 °C
?. [Lysing Bacterial Cells]
Add  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.
20
?. [Lysing Bacterial Cells]
Add  of enzymatic lysis buffer (ELB) + Enzyme to sample tube.Resuspend bacterial cell pellet by vortexing.
180 µl
?. [Lysing Bacterial Cells]
Incubate at  for  .
56 °C
?. [Lysing Bacterial Cells]
Add  of Proteinase K. Add  of Buffer AL. Vortex on high setting for 5 seconds to mix.
25 µl
200 µl
?. [Lysing Bacterial Cells]
Incubate at  for at least
56 °C
?. [Lysing Bacterial Cells]
Add  of 100% Ethanol and mix by vortexing.
200 µl
?. [Lysing Bacterial Cells]
Add  of Proteinase K. Resuspend bacterial cell pellet by vortexing.
20 µl
?. [Lysing Bacterial Cells]
Incubate at  for
56 °C
?. [Lysing Bacterial Cells]
Add   of RNase A. Mix by vortexing and incubate at room temperature for
4 µl
?. [Lysing Bacterial Cells]
Add  of Buffer AL and mix by vortexing. (Check for precipitate in AL, if precipitate is present, warm to 56oC until it is fully dissolved)
200 µl
?. [Lysing Bacterial Cells]
Add of 100% ethanol and mix by vortexing.
200 µl
?. [Lysing Bacterial Cells]
Add   of RNase A. Mix by vortexing and incubate at room temperature for
4 µl
?. [Collect Cells]
Removed the inoculated TSB samples from the incubator.