TITLE: Long-read DNA preparation for bacterial isolates.
AUTHORS: He Sun, Christian Brandt, Anna Schnürer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">General:</span></div><div class = "text-block">This is an optimized DNA isolation protocol adapted to the properties of bacteria isolated from biogas substrate and digestate, using NucleoBond® AXG Columns and NucleoBond® Buffer Set. The protocol reliably retrieves DNA of sufficient quality, length and yield. The performance has been validated on seven bacterial isolates from one biogas reactor and one biogas plant. Buffers and proteinase K can be found in NucleoBond Buffer Set III.</div><div class = "text-block"><span style = "font-weight:bold;">Observed Performance on seven isolates runs using this protocol:</span></div><div class = "text-block">N50 Ranges between 22,000 and 46,000 bp. General throughput is between 16-22 Gigabases of a single flowcell (flowcell 106 Rev D, using the LSK-109 Kit). </div><div class = "text-block"><span style = "font-weight:bold;">Funding:</span></div><div class = "text-block">This work was funded by the China Scholarship Council (CSC) [Grant No. 201606350190] and SLU.</div></div>

[STEPS]
?. [Samples preparation]
Add cation-adjusted Müller-Hinton broth in a 15 ml Falcon tube. (Müller-Hinton medium is used here for antibiotic-resistant bacteria)
[CAMHB]
?. [Samples preparation]
Add 1/4 10 µl-loop of colony from an agar plate in the solution.
?. [Samples preparation]
Vortex the mixture for 5 s at maximum speed.
?. [Samples preparation]
Incubate the mixture overnight on a shaking bench at the speed of 120 rpm at 37 ℃.
37 °C
?. [Cell disruption]
Add 2 ml of the incubated mixture in a 2.5 ml Eppendorf tube.
?. [Cell disruption]
Centrifuge the mixture.
20 °C
?. [Cell disruption]
Resuspend the bacterial pellet in Buffer G3 by vortexing.
[Buffer G3]
?. [Cell disruption]
Add lysozyme and Proteinase K
[lysozyme (100 mg/ml)]
[Proteinase K]
?. [Cell disruption]
Gently resuspend the mixture and incubate at 37 ℃ for 20 min without shaking.
37 °C
?. [Cell disruption]
Add Buffer G4
[Buffer G4]
?. [Cell disruption]
Gently resuspend the mixture and incubate at 50 ℃ for 30 min without shaking.
50 °C
?. [Cell disruption]
Centrifuge the mixture.
20 °C
?. [Equilibration]
Equilibrate the column (AXG 20) with Buffer N2.
[Buffer N2]
?. [Binding]
Add Buffer N2 (room temperature) to the sample. Vortex the mixture for 15 s at maximum speed.
[Buffer N2]
?. [Binding]
Load 1 ml of the mixture on the column. Allow it to enter the resin by gravity flow.
?. [Cell disruption]
Discard the supernatant. (For anaerobes, due to low concentration of bacterial culture, it is suggested to repeat step 5 to step 7 several times in order to obtain enough amount of DNA)
?. [Binding]
Reload 1 ml of the mixture on the column. Do not reload if the mixture of former step has not been filtered completely in case the column gets clogged.
?. [Binding]
Reload the rest mixture.
?. [Cell disruption]
Transfer the supernatant to a 5 ml Eppendorf tube.
?. [Wash]
Wash the column with Buffer N3.
[Buffer N3   three times]
?. [Elution]
Elute the genomic DNA with Buffer N5. Collect the DNA solution in a 2.5 ml Eppendorf tube.
[Buffer N5]
The average concentration of DNA is approximately 10 µg/ml. Store at 4 ℃ if you want to stop here
?. [Cleaning & condensing]
Add magnetic Beads ( 0.35 volume, AMPure) to the DNA sample. Gently resuspend by hand.
[Magnetic beads]
[DNA sample]
?. [Cleaning & condensing]
Incubate the mixture.
[Room temperature]
?. [Cleaning & condensing]
Spin down and let the beads bind to magnetic plate until the liquid is clear.
?. [Cleaning & condensing]
Stay on the magnet and remove supernatant.
?. [Cleaning & condensing]
Wash with Ethanol. Do not disturb pellet.
[Ethanol (80%)]
?. [Cleaning & condensing]
Remove supernatant.
?. [Cleaning & condensing]
[Redo the washing once]
?. [Cleaning & condensing]
Spin down and let the beads bind to magnetic plate again.Remove the residual liquid with a 10 µl pipette.
?. [Cleaning & condensing]
Add nuclease free water and resuspend gently.
[Nuclease free water]
?. [Cleaning & condensing]
Incubate the DNA solution.
37 °C
?. [Cleaning & condensing]
Spin down and put on the magnetic plate.Retrieve the supernatant (DNA) with a 10 µl pipette.
Determine dsDNA concentration e.g. by Qubit.Determine DNA length by Agarose gel or Bioanalyzer.