TITLE: DOH Workshop Protocol Part 1: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria  (MagAttract HMW DNA)
AUTHORS: Vesa Qarkaxhija, Bryan Wee, Natalie Ring

[DESCRIPTION]
This protocol enables extraction of high-molecular-weight DNA from gram-negative bacterial cultures.

[BEFORE_START]
If precipitate is formed in Buffer ATL, dissolve by incubating at 37°C with occasional shaking. The presence of precipitate can result in incomplete lysis.
Prepare (but do not start) the thermomixer to 56°C at 900rpm for 30min for the lysis step.

[GUIDELINES]
Important points throughout protocol:

 

 


 



[STEPS]
SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
1. Pellet 1.5 mL of the Gram negative bacterial cell culture by spinning at 5000 x g for 3 min. 

 

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
2. Remove and discard supernatant without disturbing the pellet. Use a P200/P20 pipette if there is a small amount of supernatant remaining. 

 

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
3. Resuspend the bacterial pellet in 180 µLBuffer ATL, add 20 µLProteinase K and mix by flicking/stirring the tube. 


 




SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
4. Incubate for 30 min on thermomixer at 56 °C shaking at 900rpm. 

Things to do while waiting:

Ensure that Buffers MW1 and PE were prepared according to instructions on them (i.e. adding appropriate amount of Ethanol).

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
5. Add 4 µLRNase A to the sample, mix by pulse vortexing or by tapping the tube several times, and incubate for2 min at Room temperature.

 


SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
6. Set the thermomixer to 21 °C to give it time to cool for next steps.

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
7. DOH Workshop Protocol Part 1Fully mix MagAttract Suspension G. Vortex the MagAttract Suspension G vigorously until thoroughly mixed ∼10 s.

 


SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
8. Add 15 µL MagAttract Suspension G and 280 µL Buffer MB to the sample. Mix by pulse vortexing (1 s-2 s quick presses x 3). 
 


SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
9. Place the tube onto the thermomixer and incubate at 21 °Cfor 3 min at 1400 rpm..

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
10. Place tubes onto magnetic rack and wait until beads pellet (∼1 min).

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
11. While leaving tubes on the magnet, carefully remove supernatant without disturbing the beads using a P1000.

 


SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
12. Remove tube from magnetic rack and add 700 µL Buffer MW1 to the tube, flick the tube until bead pellet is unstuck from tube wall.


 


SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
13. Place the tube onto the thermomixer and incubate at 21 °C for 3 min at 1400 rpm.

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
14. Place tubes onto magnetic rack and wait until beads pellet (∼1 min).

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
15. While leaving tubes on the magnet, carefully remove supernatant without disturbing the beads using a P1000.


 


SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
16. For the second wash, remove tube from magnetic rack and add 700 µL Buffer MW1 to the tube, flick the tube until bead pellet is unstuck from tube wall.


 

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
17. Place the tube onto the thermomixer and incubate at 21 °C for 3 min at 1400 rpm.

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
18. Place tubes onto magnetic rack and wait until beads pellet (∼1 min).

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
19. While leaving tubes on the magnet, carefully remove supernatant without disturbing the beads using a P1000.


 



SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
20. Remove tube from magnetic rack and add 700 µLBuffer PE to the tube, flick the tube until bead pellet is unstuck from tube wall.

PE Wash 1
SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
21. Place the tube onto the thermomixer and incubate at 21 °C for 3 minat 1400 rpm..

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
22. Place tubes onto magnetic rack and wait until beads pellet (∼1 min).

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
23. While leaving tubes on the magnet, carefully remove supernatant without disturbing the beads using a P1000.


 


SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
24. For the second wash, remove tube from magnetic rack and add 700 µLBuffer PE to the tube, flick the tube until bead pellet is unstuck from tube wall.

PE Wash 2

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
25. Place the tube onto the thermomixer and incubate at 21 °C for 3 min at 1400 rpm.

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
26. Place tubes onto magnetic rack and wait until beads pellet (∼1 min).

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
27. While leaving tubes on the magnet, carefully remove supernatant without disturbing the beads using a P1000. Then use a P200 to remove any traces of Buffer PE.


 



SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
28. While keeping the tube on the magnetic rack, slowly pipette 700 µLdistilled water by pipetting down the side of the tube opposite the pellet, without disturbing any of the beads/pellet. Leave for 30 s-1 min and then remove the supernatant without disturbing any of the beads/pellet. 


 




SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
29. Repeat step 28.

 



SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
30. Remove the tube from the magnetic rack and add50 µL of nuclease free water. Resuspend the beads by slowly pipetting or stirring with the pipette tip. 

 




SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
31. Place the tube onto the thermomixer and incubate at t 21 °Cfor 3 min at 1400 rpm.

SECTION: Purification of High- Molecular Weight Genomic DNA from Gram-Negative Bacteria
32. Pellet beads on magnet and slowly pipette DNA eluate into a new lo-bind tube.