TITLE: C-SOP-101: Bacterial Genomic DNA Isolation using the Nexttec 1-step Kit (96-plate format)
AUTHORS: Mihir Kekre

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes an easy, convenient and rapid method of genomic DNA isolation and purification from Gram (+) and (-) bacterial broth suspensions. The workflow, dubbed as a 1-step, single buffer system is a reversal of the traditional silica membrane and magnetic bead-based DNA extraction protocols. Proteins, cellular debris and other low-molecular weight contaminants are retained by the nexttec™ sorbent while pure dsDNA is pulled through as an eluate in a single wash step.</div><div class = "text-block">The protocol described is suitable for extraction of DNA from pure bacterial isolates of the following organisms:</div><div class = "text-block"><span>1. </span><span style = "font-style:italic;">Acinetobacter baumannii</span></div><div class = "text-block"><span>2. </span><span style = "font-style:italic;">Escherichia coli</span></div><div class = "text-block"><span>3. </span><span style = "font-style:italic;">Klebsiella pneumoniae</span></div><div class = "text-block"><span>4. </span><span style = "font-style:italic;">Staphylococcus aureus</span></div><div class = "text-block"><span>5. </span><span style = "font-style:italic;">Neisseria gonorrhoea</span></div><div class = "text-block"><span>6. </span><span style = "font-style:italic;">Salmonella typhi and typhimurium</span></div><div class = "text-block"><span>7. </span><span style = "font-style:italic;">Non-typhoidal Salmonella spp.</span></div><div class = "text-block"><span>8. </span><span style = "font-style:italic;">Pseudomonas aeruginosa</span></div><div class = "text-block"><span>9.</span><span style = "font-style:italic;"> Streptococcus pneumoniae</span></div><div class = "text-block"><span>10. </span><span style = "font-style:italic;">Vibrio cholerae</span></div><div class = "text-block">This protocol has been adapted from nucleic acid extraction methods developed by nexttec Biotechnologie GmbH.</div><div class = "text-block"><span>This is an open-access protocol distributed under the terms of the </span><a href="https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Creative Commons Attribution-NonCommercial-ShareAlike</span></a><span style = ":;">.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://protocols-files.s3.amazonaws.com/files/cuzrbf8zp.png?X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAJYFAX46LHRVQMGOA%2F20210115%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20210115T144235Z&X-Amz-SignedHeaders=host&X-Amz-Expires=604800&X-Amz-Signature=4d30786ca7aacf846ebb45f629340425a877a4d64c2540df606a3635695c861e" /></div></div>

[STEPS]
?. [Before Starting]
Create an organised bench space by clearing away all clutter in order to maximize work efficiency. Avoid movements that will expose sterile materials to airborne contaminants.
?. [Preparation of the Overnight Bacterial Suspension]
Refer step 5 for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella Typhi, Non-typhoidal Salmonella spp., Pseudomonas aeruginosa, and Vibrio cholerae.Refer step 6 for Neisseria gonorrhoea.Refer step 7 for Streptococcus pneumoniae.
?. [Before Starting]
Treat all isolates and cultures as infectious. Local laboratory safety practices should be followed at all times.
?. [Preparation of the Overnight Bacterial Suspension]
Broth suspension preparation for: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella Typhi, Non-typhoidal Salmonella spp., Pseudomonas aeruginosa, and Vibrio cholerae.
?. [Preparation of the Overnight Bacterial Suspension]
Broth suspension preparation for: Neisseria gonorrhoea.
?. [Preparation of the Overnight Bacterial Suspension]
Broth suspension preparation for: Streptococcus pneumoniae.
?. [Preparation of the Overnight Bacterial Suspension]
Grow the bacterial culture on Blood Agar medium and incubate the plates for  at .
37 °C
?. [Preparation of the Overnight Bacterial Suspension]
After incubation, examine each plate for contamination to ensure purity of isolates/colonies.
?. [Preparation of the Overnight Bacterial Suspension]
Pick a pure, single bacterial colony off the agar plate and passage to grow in Tryptic Soy Broth (TSB) at  in dry incubator for . Next, proceed to pelleting (step 8).
35 °C
?. [Preparation of the Overnight Bacterial Suspension]
Grow the bacterial culture on Chocolate Agar medium and incubate the plates for  at  in a CO2 controlled incubator or shift candle jar.
35 °C
?. [Preparation of the Overnight Bacterial Suspension]
After incubation, examine each plate for contamination to ensure purity of isolates/colonies.
?. [Preparation of the Overnight Bacterial Suspension]
For each sample to be extracted, dispense  of Alkaline Peptone Water (APW) in a sterile 5 mL tube.
2.5 mL
?. [Preparation of the Overnight Bacterial Suspension]
Using a sterile, autoclaved toothpick, pick a pure, isolated colony of N. gonorrhoea. Immediately suspend and emulsify the colony scoop on the inner wall of the tube containing APW in order to break the cell clumps. Mix properly until a homogeneous bacterial suspension is obtained.
?. [Preparation of the Overnight Bacterial Suspension]
Measure this suspension using a McFarland nephelometer. Adjust and remeasure until the homogenous bacterial suspension standard (turbidity) is 3.0.Next, proceed to pelleting (step 8).
?. [Preparation of the Overnight Bacterial Suspension]
Grow the bacterial culture on Sheep Blood Agar medium and incubate the plates for  at  in a CO2 controlled incubator or shift candle jar.
36 °C
?. [Preparation of the Overnight Bacterial Suspension]
After incubation, examine each plate for contamination to ensure purity of isolates/colonies.
?. [Preparation of the Overnight Bacterial Suspension]
For each sample to be extracted, dispense  of 0.85% Normal Saline Solution (NSS) in a sterile 5 mL tube.
2.5 mL
?. [Preparation of the Overnight Bacterial Suspension]
Using a sterile, autoclaved toothpick, pick a pure, isolated colony of S. pneumoniae. Immediately suspend and emulsify the colony scoop on the inner wall of the tube containing NSS in order to break the cell clumps. Mix properly until a homogeneous bacterial suspension is obtained.
?. [Preparation of the Overnight Bacterial Suspension]
Measure this suspension using a McFarland nephelometer. Adjust and remeasure until the homogenous bacterial suspension standard (turbidity) is 3.0.Next, proceed to pelleting (step 8).
?. [Pelleting the Bacterial Culture]
Carefully transfer  of each broth suspension culture (with a minimum OD600 = 1.5) grown overnight to an empty well of a clean, sterile round-bottom 96-deepwell plate.
1.5 mL
Ensure that pipette tips are lowered to the bottom of each well when dispensing broth into it. This will avoid any cross-well droplet spillage and, in turn, sample contamination.
?. [Pelleting the Bacterial Culture]
Repeat step 8 for each of the isolates from which DNA needs to be isolated and note down the positional layout of all samples on the deepwell plate.
The 96-deepwell plate has wells labelled A1 to H12. It is recommended to fill only up to 95 positions (i.e. A1 to G12) leaving H12 empty as a control well.
?. [Pelleting the Bacterial Culture]
Seal the plate tight with an 'Alu' seal (provided with the Nexttec kit) and centrifuge the suspensions at .
Centrifuge: 3000 33, 4 min
?. [Pelleting the Bacterial Culture]
Carefully remove the plate from the centrifuge and discard the supernatant from each well by aspirating the liquid using a multichannel pipette.
Take extra care not to dislodge any pellets.
?. [Pelleting the Bacterial Culture]
Invert the plate onto a clean piece of tissue to absorb any excess supernatant drain-off. Give the plate a few gentle taps to ensure all of the liquid has drained onto the paper.
?. [Cellular Lysis of the Bacterial Pellet]
Pre-heat the incubator-shaker to  and set the rotation speed at .
56 °C
33
?. [Cellular Lysis of the Bacterial Pellet]
Defrost all of the frozen reagents, and place them along with the chilled components in a large bucket of ice.
For best results, ensure the larger volume contents are fully submerged in the ice.
?. [Cellular Lysis of the Bacterial Pellet]
Gather all of the lyophilised Lysozyme powder at the bottom of the tube by centrifuging the vial in the tabletop tube spinner.To prepare  of Lysozyme solution, add the relevant volume of nuclease-free water to the lyophilised lysozyme vial provided.Kit 20N.901 – add  of waterKit 20N.902 – add  of waterKit 20N.904 – add of waterKit 20N.924 – add  of water
1.5 mL
4.5 mL
6 mL
30 mL
?. [Cellular Lysis of the Bacterial Pellet]
Using Table 1 below,  in a 50 mL Falcon tube, prepare Lysis Mix 1 (LM1) mixing together the required volumes of: Buffer B, Lysozyme or Lysostaphin solution and RNase A.
i. Always prepare fresh LM1 for each experiment; Do NOT store it overnight.ii. Prepare a separate LM1 for Gram (-) and Gram (+) samples.
?. [Cellular Lysis of the Bacterial Pellet]
Using a P200 pipette (single or multi-channel), add: to every well containing a Gram (-) organism. to every well containing a Gram (+) organism.
120 µl
135 µl
?. [Cellular Lysis of the Bacterial Pellet]
Gently re-suspend the pelleted cells by pipetting the LM1 up and down.
Pipette extremely slowly to avoid frothing of the mixture.
?. [Cellular Lysis of the Bacterial Pellet]
Carefully raise the plate above eye level (without tilting) and check that to confirm that the pelleted clumps at the well bottom have fully dissolved in LM1.
?. [Cellular Lysis of the Bacterial Pellet]
Seal the deepwell plate tightly using an 'Alu' seal.
Ensure the seal is fully affixed by flattening the center and edges with a roller.
?. [Cellular Lysis of the Bacterial Pellet]
Place the plate in the incubator-shaker at ,  for .
56 °C
33
?. [Cellular Lysis of the Bacterial Pellet]
When the incubation is complete, remove the plate and briefly centrifuge at  to gather any condensed droplets from underneath the seal.Now, preset the incubator-shaker to  and .
Centrifuge: 500 33, 10 s
68 °C
33
?. [Cellular Lysis of the Bacterial Pellet]
Back at the bench, gripping the plate tightly with one hand, carefully peel back the 'Alu' seal and discard it.
?. [Cellular Lysis of the Bacterial Pellet]
Using Table 2 below,  in a 50 mL Falcon tube, prepare Lysis Mix 2 (LM2) mixing together the required volumes of: SDS solution, Proteinase K, DTT and EDTA.
i. Always prepare fresh LM2 for each experiment; Do NOT store it overnight.ii. There are no separate LM2 for Gram (-) and Gram (+) samples.
?. [Cellular Lysis of the Bacterial Pellet]
Using a P200 pipette (single or multi-channel), add  of LM2 to every sample well of the deepwell plate.Mix the solution repeatedly by pipetting up and down until homogenous.
120 µl
Pipette extremely slowly to avoid frothing of the mixture.
?. [Cellular Lysis of the Bacterial Pellet]
Reseal the plate and return it to the incubator-shaker at now set to ,  for .
68 °C
33
i. Longer incubation times lead to more efficient cellular lysis. Lysates can be incubated up to 6 hours at this temperature (with a mandatory minimum of 3.5 hours). If the starting suspensions were ‘stringy and goopy’ due to the presence of thick capsular formations or biofilms, increase the lysis time by 1-2 hours. Clumpy final lysates can clog the extraction column and drastically reduce DNA yield.ii. Shaking at 230 rpm is critical to homogenising the lysis mix through dissolution of course cellular debris. iii. The protocol can be paused on completion of the 4 hour incubation period. Pauses should only be taken if absolutely necessary as results can vary slightly.The deepwell plate with lysates needs to be centrifuged briefly to gather condensation, resealed with a fresh Alu seal and stored at    .
?. [Before Starting]
Prior to initiating the protocol, ensure that all active workbenches are cleaned with 80% ethanol, all relevant personal protective clothing is worn and the work area is prepared for DNA extraction according to local GLP guidelines.
?. [Equilibration of the cleanPlate96]
Place a cleanPlate 96 with fresh, unused columns flat on the workbench (with a waste plate set up underneath it).
?. [Cellular Lysis of the Bacterial Pellet]
20 minutes before the incubation is due to finish, start equilibrating the 96-column plate as described in the next section (steps 28-32).
?. [Equilibration of the cleanPlate96]
Pour out Prep Solution into a reservoir. Using the P300/P1000 8-channel pipette (with filtered tips), add  of ice-cold Prep Solution into each column that is to be used for DNA isolation.
350 µl
Equilibrate only those wells/columns corresponding to occupied sample wells on the 96-deepwell (i.e. those that will be needed for DNA isolation)Do NOT engage the entire plate if you wish to use less than 96 sample wells.
When expurgating the Prep solution into each well, ensure that: i. the Prep Solution is ice cold.ii. the pipette tips are held exactly perpendicular and upright above the centre of the column.iii. they are just above the surface of the white resin WITHOUT touching it. If the tips come in contact with the resin, it may rupture.
?. [Equilibration of the cleanPlate96]
Incubate for  at .
68 Room temperature
?. [Equilibration of the cleanPlate96]
Centrifuge the plate setup at . Once complete, remove the plate setup from the centrifuge and discard the waste-plate below.
Centrifuge: 1300 33, 1 min
i. You can use either a previously spent or a fresh, unwrapped cleanPlate96 to balance inside the centrifuge.ii. For batches less than 96 samples (or 96 occupied wells), the waste plate can be reused for this step until every column on the cleanPlate 96 has been utilised.
?. [Equilibration of the cleanPlate96]
Place the cleanPlate96 onto a new, fresh deep-well plate (provided with the kit). Label this the DNA collection plate.
Continue with step 33 soon after completing the above. If the columns wetted with Prep solution are allowed to dry out for too long, DNA yield will reduce drastically.
?. [DNA Purification and Elution]
Remove the lysate 96-plate (from step 26) from the incubator. Extra care should be taken when handling it as the plate will be hot.
When resuming a protocol pause, the lysates (stored at 4°C overnight) should be left to warm up to   for   prior to step 34.
?. [DNA Purification and Elution]
Briefly centrifuge the plate to gather any condensation on the seal.Slowly and carefully peel off the seal avoiding any droplet spillage. Gently pipette up and down 10 times to homogenise the lysates.
Pipette extremely slowly to avoid frothing of the mixture.
?. [DNA Purification and Elution]
Using a P300/P1000 8-channel pipette (with filtered tips),  transfer  of the lysate to the corresponding positions on the cleanPlate96.Reseal the deepwell plate and store the remaining lysate at .
120 µl
4 °C
i. Using a permanent marker, outline the wells whose columns you wish to use for this batch of extractions.ii. The remaining lysate is only viable for up to 36 hours at 4°C. Results may vary from fresh lysate.iii. When expurgating the lysates into the well, ensure that: a) the pipette tips are held exactly perpendicular and upright above the centre of the column.b) the pipette tips are just above the surface of the white resin without touching it. c) the entire lysate from the tip is deposited onto the column without bubbles.
?. [DNA Purification and Elution]
Incubate for  at .
68 Room temperature
?. [DNA Purification and Elution]
Centrifuge the cleanPlate96 at .
Centrifuge: 1900 33, 90 s, 4 10
Do NOT exceed the spin speed specified for this step. Operating at >1900rpm will cause proteins and cellular debris to elute through and contaminate the DNA.
?. [DNA Purification and Elution]
The DNA collection plate now contains purified double-stranded genomic DNA.
Store the plate setup (including the used cleanPlate 96) at 4°C until quantification assays have shown adequate amounts in solution.In case there is insufficient DNA, try either of the following remedial measures:1. Add a further 120 µL of Prep solution to the same column wells and repeat steps 36-37.OR2. Repeat steps 35-37 on a fresh set of cleanPlate 96 column positions with the remaining lysate from step 35.
?. [Additional Information & Troubleshooting]
Base Protocol and User Manual