TITLE: Isolation of HMW gDNA from a bioreactor biofilm for long read sequencing 
AUTHORS: Viola A Manning, Kristin Trippe

[DESCRIPTION]
This protocol describes isolation of HMW DNA for long read sequencing from a bioreactor biofilm. 

[STEPS]
SECTION: Storing bioreactor biofilm
1. Aliquot 750 uL of biofilm into 1.5 mL eppendorf tubes. 

SECTION: Storing bioreactor biofilm
2. Spin in microcentrifuge (eppendorf 5424) at ~21,000 x g for 5 minutes.5 min 

SECTION: Storing bioreactor biofilm
3. Resuspend pellet in 1 mL   by pipetting up and down multiple times. 

SECTION: Storing bioreactor biofilm
4. Store resuspended pellet at -80 °C  until ready to process.

SECTION: Disrupting the biofilm
5. Transfer 1 mL of sample into 2mm bashing beads (Zymo S6003)
 

SECTION: Disrupting the biofilm
6. Place on Vortex Genie with horizontal adapter and shake for 10 minutes, top speed.
SECTION: Disrupting the biofilm
7. Remove supernatant to a 2 mL tube.
SECTION: Disrupting the biofilm
8. Centrifuge 5000 x g for 1 minute.1 min 

SECTION: Extract the pellet
9. Remove supernatant to a 5 mL eppendorf tube.
SECTION: Extract the pellet
10. Resuspend pellet in 500 uL PBS and pipette mix until pellet is visibly resuspended.
SECTION: Extract the pellet
11. The remaining steps of this protocol were adapted from the Microbial Lysis and DNA Purification section of   .

Centrifuge at 5000 x g for 1 minute and combine supernatant with the original supernatant. 1 min 

SECTION: Extract the pellet
12. Resuspend pellet in 1 mL PBS, spin, and discard supernatant.
SECTION: Extract the pellet
13. Add 500 uL TE and 125 uL 100 mg/ml lysozyme in TE.
SECTION: Extract the pellet
14. Pipette mix until pellet is visibly resuspended and incubate at 55 °C  for 30 minutes, flicking occasionally.30 min 

SECTION: Extract the pellet
15. Combine with supernatants and aliquot ~ 1 mL into each of two 2 mL eppendorf tubes.
SECTION: Extract the pellet
16. To each tube, add 50 uL 10% SDS and 25 uL Proteinase K, pipette mix, incubate at 55 °C  for 30 minutes on a Thermomixer at 500 rpm. Incubate longer if it looks like lysis is incomplete.30 min 

SECTION: Extract the pellet
17. Centrifuge 5000 x g for 1 minute and transfer 1 mL supernatant into two each 5 mL eppendorf tubes.1 min 

SECTION: Extract the pellet
18. Add 2 mL Quick-DNA MagBinding Buffer.
SECTION: Extract the pellet
19. Add 66 uL well resuspended magnetic beads, mix by pipetting up and down 5 times, put on rotating mixer for 10 minutes.10 min 

SECTION: Extract the pellet
20. Transfer tube to magnetic stand, let beads separate, remove and discard supernatant.

SECTION: Extract the pellet
21. Remove from the magnetic stand, add 500 uL Quick-DNA MagBinding Buffer, transfer to 2 mL eppendorf tube.
SECTION: Extract the pellet
22. Pipette 5 times to mix and then put on rotator for 5 minutes. 5 min 

SECTION: Extract the pellet
23. Place on magnetic stand and remove supernatant.
SECTION: Extract the pellet
24. Remove from magnetic stand, add 500 uL Pre-Wash Buffer, and pipette 10 times to reususpend.
SECTION: Extract the pellet
25. Place on magnetic stand, let beads separate, and remove supernatant. 
SECTION: Extract the pellet
27. Transfer to clean 2 mL Eppendorf tube, place on stand, let beads separate, and remove supernatant. 
SECTION: Extract the pellet
28. Repeat steps 26 and 27.
SECTION: Extract the pellet
29. Dry beads in hood for 20 minutes.
SECTION: Extract the pellet
30. Resuspend in 100 uL DNA elution buffer, pipette mix 20 times, and incubate on Thermomixer for 5 min at 25 °C  while shaking at 1000 rpm.5 min 

SECTION: Extract the pellet
31. Transfer sample to magnetic stand, transfer solution to new tube.
SECTION: Extract the pellet
32. Store at  -20 °C.

SECTION: Extract the pellet
26. Remove from stand, and add 900 uL g-DNA wash buffer and pipette 10 times to resuspend.