TITLE: MagAttract + Metapolyzyme metagenomic gDNA extraction from urine
AUTHORS: Natalie Ring

[DESCRIPTION]
A protocol for the metagenomic extraction of bacterial DNA from urine samples (optimised using dog urine), for use in a rapid diagnostics pipeline. At the end of the protocol, the DNA is cleaned up and ready for rapid barcoding (SQK-RBK004) library preparation for nanopore sequencing (or whatever other application you want to do). 

Unless otherwise stated, all reagents should be included in the listed kits.

[BEFORE_START]
"Buffer P1" is required for the metapolyzyme lysis incubation: 50 mM Tris, 10 mM EDTA, pH 8.0
Metapolyzyme is used here at a concentration of 3.3 mg/ml (resuspend 5 mg lyophilized powder in 1.5 ml PBS pH 7.5) 
We recommend using low DNA-binding tubes throughout, but definitely for the elution/storage of DNA


[GUIDELINES]
This protocol, an adaptation of Qiagen's MagAttract HMW DNA kit, was developed by Natalie Ring and Alison Low for the Dogstails project, a collaboration between the Roslin Institute and the Royal (Dick) School of Veterinary Studies funded by the Dogs Trust. We are grateful to the dogs (and their owners) who donated samples to the R(D)SVS's Hospital for Small Animals, many of which were used in the development of this protocol.

Please follow on Twitter for latest updates, papers and results:

@NatalieAnneRing



[STEPS]
SECTION: Extended pre-lysis spin down
1. Pellet 2x 1.5 ml aliquots of urine in 1.5 ml tubes by centrifuging at maximum speed (~13,000 RPM/16,000 xg) for 20 minutes, then discard supernatant

3 mL 


16,000 x g, 20 min, RT Room temperature 


 


SECTION: Metapolyzyme &amp; Proteinase K Lysis
2. Resuspend cell pellets (which might be invisible) and combine in 160 µl buffer P1 (50 mM Tris, 10 mM EDTA, pH 8.0)

160 µL 

SECTION: Metapolyzyme &amp; Proteinase K Lysis
3. Add 20 µl metapolyzyme (3.3 mg/ml, 5 mg resuspended in 1500 µl PBS) and mix by flicking the tube

20 µL 

SECTION: Metapolyzyme &amp; Proteinase K Lysis
4. Incubate on a thermomixer for 60 minutes at 37°C with 900 RPM shaking

  



SECTION: Metapolyzyme &amp; Proteinase K Lysis
5. Add 20 µl MagAttract proteinase K and mix by flicking the tube

20 µL 

SECTION: Metapolyzyme &amp; Proteinase K Lysis
6. Incubate on a thermomixer for 30 minutes at 56°C with 900 RPM shaking

  



SECTION: MagAttract DNA isolation and washing
7. Add 150 µl MagAttract buffer AL and mix by pulse vortexing

150 µL 


 

SECTION: MagAttract DNA isolation and washing
8. Add 15 µl MagAttract Suspension G and 280 µl MagAttract buffer MB and mix by pulse vortexing

15 µL 
280 µL 

 



SECTION: MagAttract DNA isolation and washing
10. Spin down briefly, then pellet beads on magnet and remove supernatant


SECTION: MagAttract DNA isolation and washing
11. Add 700 µl MagAttract buffer MW1 and incubate on a thermomixer for 1 minute at room temperature with 1,400 RPM shaking

700 µL 
  




SECTION: MagAttract DNA isolation and washing
12. Repeat steps 10 and 11
SECTION: MagAttract DNA isolation and washing
13. Spin down briefly, then pellet beads on magnet and remove supernatant



SECTION: MagAttract DNA isolation and washing
14. Add 700 µl MagAttract buffer PE and incubate on a thermomixer for 1 minute at room temperature with 1,400 RPM shaking

700 µL 
  


SECTION: MagAttract DNA isolation and washing
15. Repeat steps 13 and 14
SECTION: MagAttract DNA isolation and washing
16. Spin down briefly, then pellet beads on magnet and remove supernatant

SECTION: MagAttract DNA isolation and washing
17. Rinse the pelleted beads on the magnetic rack with 700 µl distilled water by pipetting down the opposite wall of the tube, then incubate for 1 minute on the magnetic rack

700 µL 

SECTION: MagAttract DNA isolation and washing
18.  Remove distilled water
SECTION: MagAttract DNA isolation and washing
21. Add 50 µl nuclease-free water off the magnet, to resuspend the bead pellet

50 µL 

SECTION: MagAttract DNA isolation and washing
22. Incubate on a thermomixer for 3 minutes at room temperature with 1,400 RPM shaking

  


SECTION: MagAttract DNA isolation and washing
23. Spin down briefly, then pellet beads on magnetic rack and keep supernatant in a low-DNA binding 1.5 mL tube (e.g. Eppendorf or Sarstedt)


SECTION: Qubit Pre-clean-up quantification
24. Quantify DNA using Qubit dsDNA HS kit. If DNA concentration is an appropriate concentration for your experiment (for us, this means at least 0.2 ng/µl), continue to clean-up steps.

  
1 µL 
199 µL 

SECTION: ProNex DNA clean-up
25. Add 150 µl room temperature ProNex beads to your entire tube of DNA (49 µl)

200 µL 


 

SECTION: ProNex DNA clean-up
26. Mix well by slowly pipetting up and down 10 times
SECTION: ProNex DNA clean-up
27. Incubate at room temperature for 10 minutes (no shaking needed)

10 min 
Room temperature 

SECTION: ProNex DNA clean-up
28. Spin down briefly, then pellet beads on magnet and remove supernatant
SECTION: ProNex DNA clean-up
29. Rinse the pelleted beads on the magnetic rack by pipetting 200 µl ProNex Wash Buffer down the opposite wall of the tube, then incubate at room temperature for 60 seconds (no shaking), then remove Wash Buffer

200 µL 
Room temperature 
1 min 

SECTION: ProNex DNA clean-up
30. Repeat step 26
SECTION: ProNex DNA clean-up
31. Air-dry (lid open) the sample on the magnetic rack for 5 minutes (longer is OK, no more than 60 minutes)

Room temperature 
5 min 

SECTION: ProNex DNA clean-up
32. Add 20 µl nuclease-free water off the magnet. Resuspend the pellet by flicking the tube, then incubate at room temperature for 5 minutes (no shaking needed)

20 µL 
Room temperature 
5 min 

SECTION: ProNex DNA clean-up
33. Spin down briefly, then pellet the beads on magnet and keep supernatant in a low DNA-binding tube
SECTION: Qubit post-clean-up quantification
34. Quantify DNA using Qubit dsDNA HS kit. If DNA concentration is an appropriate concentration for your experiment (for us, this means at least 0.2 ng/µl), continue to library preparation.

  
1 µL 
199 µL 

SECTION: MagAttract DNA isolation and washing
9. Incubate on a thermomixer for 3 minutes at room temperature with 1,400 RPM shaking

  

SECTION: MagAttract DNA isolation and washing
19. Repeat steps 17 and 18
SECTION: MagAttract DNA isolation and washing
20. Spin down briefly, then pellet beads on magnet and remove any remaining supernatant