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

[DESCRIPTION]
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.

The protocol described is suitable for extraction of DNA from pure bacterial isolates of the following organisms:
1. Acinetobacter baumannii
2. Escherichia coli
3. Klebsiella pneumoniae
4. Staphylococcus aureus
5. Neisseria gonorrhoea
6. Salmonella typhi and typhimurium
7. Non-typhoidal Salmonella spp.
8. Pseudomonas aeruginosa
9. Streptococcus pneumoniae
10. Vibrio cholerae

This protocol has been adapted from nucleic acid extraction methods developed by nexttec Biotechnologie GmbH.

This is an open-access protocol distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike.
  


[GUIDELINES]
Storage (Nexttec 1-step):
During shipment (10-15 days in transit), all kit components are stable at room temperature. 
On arrival, store: 
1. Prep Solution, Buffer B, EDTA, RNase A and Proteinase K at +2 °C to +8 °C. 
2. DTT and Lysozyme/Lysostaphin at -18 °C to -25 °C. 
3. SDS Solution, the nexttecTM cleanPlates96 and plate seals at room temperature (+20 °C to +25 °C). 
If properly stored, the expiration date on the kit will apply.

[STEPS]
SECTION: Before Starting
2. 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. 
SECTION: Preparation of the Overnight Bacterial Suspension
4. 
 

SECTION: Before Starting
3. Treat all isolates and cultures as infectious. Local laboratory safety practices should be followed at all times.
SECTION: Preparation of the Overnight Bacterial Suspension
5. Broth suspension preparation for: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella Typhi, Non-typhoidal Salmonella spp., Pseudomonas aeruginosa, and Vibrio cholerae.
SECTION: Preparation of the Overnight Bacterial Suspension
6. Broth suspension preparation for: Neisseria gonorrhoea.
SECTION: Preparation of the Overnight Bacterial Suspension
7. Broth suspension preparation for: Streptococcus pneumoniae.
SECTION: Preparation of the Overnight Bacterial Suspension
5.1. Grow the bacterial culture on Blood Agar medium and incubate the plates for 1380 min at 37 °C.

SECTION: Preparation of the Overnight Bacterial Suspension
5.2. After incubation, examine each plate for contamination to ensure purity of isolates/colonies.
SECTION: Preparation of the Overnight Bacterial Suspension
5.3. Pick a pure, single bacterial colony off the agar plate and passage to grow in Tryptic Soy Broth (TSB) at 35 °C in dry incubator for 1080 min. 

Next, proceed to pelleting (step 8).
SECTION: Preparation of the Overnight Bacterial Suspension
6.1. Grow the bacterial culture on Chocolate Agar medium and incubate the plates for 1440 min at 35 °C in a CO2 controlled incubator or shift candle jar. 

SECTION: Preparation of the Overnight Bacterial Suspension
6.2. After incubation, examine each plate for contamination to ensure purity of isolates/colonies. 
SECTION: Preparation of the Overnight Bacterial Suspension
6.3. For each sample to be extracted, dispense 2.5 mL of Alkaline Peptone Water (APW) in a sterile 5 mL tube. 

SECTION: Preparation of the Overnight Bacterial Suspension
6.4. 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.
SECTION: Preparation of the Overnight Bacterial Suspension
6.5. 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).
SECTION: Preparation of the Overnight Bacterial Suspension
7.1. Grow the bacterial culture on Sheep Blood Agar medium and incubate the plates for 1080 min at 36 °C in a CO2 controlled incubator or shift candle jar.

SECTION: Preparation of the Overnight Bacterial Suspension
7.2. After incubation, examine each plate for contamination to ensure purity of isolates/colonies. 


SECTION: Preparation of the Overnight Bacterial Suspension
7.3. For each sample to be extracted, dispense 2.5 mL of 0.85% Normal Saline Solution (NSS) in a sterile 5 mL tube. 

SECTION: Preparation of the Overnight Bacterial Suspension
7.4. 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.
SECTION: Preparation of the Overnight Bacterial Suspension
7.5. 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).
SECTION: Pelleting the Bacterial Culture
8. Carefully transfer 1.5 mL 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.
 


SECTION: Pelleting the Bacterial Culture
9. 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.
 

SECTION: Pelleting the Bacterial Culture
10. Seal the plate tight with an 'Alu' seal (provided with the Nexttec kit) and centrifuge the suspensions at 3000 rpm, 4 min. 

SECTION: Pelleting the Bacterial Culture
11. Carefully remove the plate from the centrifuge and discard the supernatant from each well by aspirating the liquid using a multichannel pipette. 
 

SECTION: Pelleting the Bacterial Culture
12. 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.
SECTION: Cellular Lysis of the Bacterial Pellet
13. Pre-heat the incubator-shaker to 56 °C and set the rotation speed at  .

SECTION: Cellular Lysis of the Bacterial Pellet
14. Defrost all of the frozen reagents, and place them along with the chilled components in a large bucket of ice. 
 

SECTION: Cellular Lysis of the Bacterial Pellet
15. Gather all of the lyophilised Lysozyme powder at the bottom of the tube by centrifuging the vial in the tabletop tube spinner.

To prepare 25 mg/mL of Lysozyme solution, add the relevant volume of nuclease-free water to the lyophilised lysozyme vial provided.
Kit 20N.901 – add 1.5 mL of water
Kit 20N.902 – add 4.5 mL of water
Kit 20N.904 – add 6.0 mLof water
Kit 20N.924 – add 30.0 mL of water




SECTION: Cellular Lysis of the Bacterial Pellet
16. 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.

   
 

SECTION: Cellular Lysis of the Bacterial Pellet
17. Using a P200 pipette (single or multi-channel), add:

120 µL to every well containing a Gram (-) organism.

135 µL to every well containing a Gram (+) organism.

SECTION: Cellular Lysis of the Bacterial Pellet
18. Gently re-suspend the pelleted cells by pipetting the LM1 up and down. 
 

SECTION: Cellular Lysis of the Bacterial Pellet
19. 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. 
SECTION: Cellular Lysis of the Bacterial Pellet
20. Seal the deepwell plate tightly using an 'Alu' seal.
 

SECTION: Cellular Lysis of the Bacterial Pellet
21. Place the plate in the incubator-shaker at 56 10,   for 40 min.

SECTION: Cellular Lysis of the Bacterial Pellet
22. When the incubation is complete, remove the plate and briefly centrifuge at 500 rpm, 10 s to gather any condensed droplets from underneath the seal.

Now, preset the incubator-shaker to 68 °C and  .

SECTION: Cellular Lysis of the Bacterial Pellet
23. Back at the bench, gripping the plate tightly with one hand, carefully peel back the 'Alu' seal and discard it.
SECTION: Cellular Lysis of the Bacterial Pellet
24. 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.

  
 

SECTION: Cellular Lysis of the Bacterial Pellet
25. Using a P200 pipette (single or multi-channel), add 120 µL of LM2 to every sample well of the deepwell plate.

Mix the solution repeatedly by pipetting up and down until homogenous.
 


SECTION: Cellular Lysis of the Bacterial Pellet
26. Reseal the plate and return it to the incubator-shaker at now set to 68 °C,   for 240 min.
 


SECTION: Before Starting
1. 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.
SECTION: Equilibration of the cleanPlate96
28. Place a cleanPlate 96 with fresh, unused columns flat on the workbench (with a waste plate set up underneath it).
SECTION: Cellular Lysis of the Bacterial Pellet
27. 20 minutes before the incubation is due to finish, start equilibrating the 96-column plate as described in the next section (steps 28-32).
SECTION: Equilibration of the cleanPlate96
29. Pour out Prep Solution into a reservoir. 

Using the P300/P1000 8-channel pipette (with filtered tips), add 350 µL of ice-cold Prep Solution into each column that is to be used for DNA isolation. 
 

 


SECTION: Equilibration of the cleanPlate96
30. Incubate for 10 min at 68 Room temperature.

SECTION: Equilibration of the cleanPlate96
31. Centrifuge the plate setup at 1300 rpm, 1 min. 

Once complete, remove the plate setup from the centrifuge and discard the waste-plate below.
 


SECTION: Equilibration of the cleanPlate96
32. Place the cleanPlate96 onto a new, fresh deep-well plate (provided with the kit). 

Label this the DNA collection plate.
 

SECTION: DNA Purification and Elution
33. 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.
 

SECTION: DNA Purification and Elution
34. 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. 
 

SECTION: DNA Purification and Elution
35. Using a P300/P1000 8-channel pipette (with filtered tips),  transfer 120 µL of the lysate to the corresponding positions on the cleanPlate96.

Reseal the deepwell plate and store the remaining lysate at 4 °C.
 


SECTION: DNA Purification and Elution
36. Incubate for 12 min at 68 Room temperature.
SECTION: DNA Purification and Elution
37. Centrifuge the cleanPlate96 at 1900 rpm, 90 s, 4 °C.
 

SECTION: DNA Purification and Elution
38. The DNA collection plate now contains purified double-stranded genomic DNA.
 

SECTION: Additional Information &amp; Troubleshooting
39. Base Protocol and User Manual