TITLE: Bacterial Genomic DNA Isolation
AUTHORS: Carly Sjogren, Carlos Goller

[DESCRIPTION]
Overview and Goals
Your bacterial isolate has been grown on agar plates. We now need to lyse open the bacteria and isolate only the genomic DNA for sequencing. For this, we need to gently lyse the bacteria with the enzyme lysozyme and then purify the genomic DNA. We will use the Monarch High Molecular Weight (HMW) DNA Extraction Kit (New England BioLabs) and protocol because we want long DNA strands to sequence with the Nanopore sequencer.

After completing this lab you will gain the following lab skills:

Lab safety and proper personal protective equipment (PPE)
Proper use of a centrifuge and pipettors
Isolation and purification of high-molecular weight genomic DNA

[BEFORE_START]
Review the figures below to learn about how the New England BioLabs Monarch High Molecular Weight (HMW) DNA Extraction kit works. 


[GUIDELINES]
Review the complete protocol before beginning. Several of the steps in this procedure are time and/or temperature sensitive, so it’s important that you know what to expect and how to manage your time.


[STEPS]
SECTION: Activity 1: Bacterial Lysis
1. Preheat two Eppendorf Thermal mixers with 1.5 mL  blocks to 37 °C  and 55 °C 

SECTION: Activity 1: Bacterial Lysis
2. Use a sterile cotton swab to obtain bacterial colonies and resuspend in 300 µL  of cold PBS or Tris buffer.

SECTION: Activity 1: Bacterial Lysis
3. Centrifuge each microtube of the sample at the maximum speed setting (>12000 x g)  for 1 min 

SECTION: Activity 1: Bacterial Lysis
3.1. Balance the centrifuge with the placement of microtubes. Use empty tubes if necessary.
SECTION: Activity 1: Bacterial Lysis
3.2. After centrifuging, there should be a “pellet” of bacteria at the bottom of the tube.
SECTION: Activity 1: Bacterial Lysis
3.3. Depending on the concentration of bacteria in the solution, this may take a few cycles of centrifuging, removing supernatant, and adding more sample to get a sufficient pellet. 
SECTION: Activity 1: Bacterial Lysis
4. Discard the supernatant. There are two methods for this depending on the strength of the pellet. 
SECTION: Activity 1: Bacterial Lysis
4.1. If the pellet is tightly packed (small clump at the bottom, little movement when swirled), you can simply dump the supernatant into the biohazard bin. The pellet will remain at the bottom. This method is commonly called "decanting the supernatant."
SECTION: Activity 1: Bacterial Lysis
4.2. If the pellet is not packed together at the bottom, first re-centrifuge up to two additional times. If it remains loose, remove the supernatant with a micropipette.  This method is commonly called "aspirating the supernatant."
 

SECTION: Activity 1: Bacterial Lysis
5. Resuspend the bacterial pellet in 300 µL  of cold PBS or Tris buffer. Add 10 µL  Lysozyme (50 mg/ml) solution and mix by vortexing for 1-2 seconds
 


SECTION: Activity 1: Bacterial Lysis
6. Add 300 µL  HMW gDNA Tissue Lysis Buffer to the sample and mix by inverting 5-10 times.

SECTION: Activity 1: Bacterial Lysis
7. Incubate at 37 °C  in a thermal mixer with agitation at    for 15 min , or until nearly clear (up to 20 min ).

 

SECTION: Activity 1: Bacterial Lysis
8. Remove the tubes after finishing incubation, and set the thermal mixer to 56 °C  (or use one already set to this temperature). 

SECTION: Activity 1: Bacterial Lysis
9. Add 20 µl of Proteinase K and mix by inverting 10–20 times.
 

SECTION: Activity 1: Bacterial Lysis
9.1. The Proteinase K is thick and will stick to the outside of the pipette tip. When pipetting, just barely break the surface so as to not end up with excess on the outside of the pipette tip. Do the same when depositing into the sample tube.
SECTION: Activity 1: Bacterial Lysis
10. Incubate at 56 °C  for 30 min in a thermal mixer at   

SECTION: Activity 1: Bacterial Lysis
11. Add 10 µL  of RNase A and mix by inverting 5–10 times. Incubate for 10 min at 56 °C  with agitation in a thermal mixer at   

SECTION: Activity 1: Bacterial Lysis
12. Add 300 µL  of Protein Separation Solution. Mix by inverting gently for 1 min 

SECTION: Activity 1: Bacterial Lysis
13. Centrifuge for 10 min at maximum rpm.
 

 


SECTION: Activity 1: Bacterial Lysis
14. If working with multiple samples, prepare and label the plastics for the upcoming steps. Each sample will require:
-Monarch Collection Tube II (not labeled)
1 Monarch Bead Retainer inserted into the collection tube; this will be used to remove the wash buffer from the gDNA bound to the beads. (labeled)
2 Monarch 2 mL  Tubes; one for phase separation and one for elution. (labeled)
1 1.5 mL  microfuge tube; this will be used to collect the eluate. (labeled)


SECTION: Activity 1: Bacterial Lysis
15. Using a 1000 µl wide-bore pipette tip, transfer the clear upper phase (containing the DNA) of each sample to a corresponding labeled Monarch 2 ml Tube. 
 

SECTION: Activity 2: HMW gDNA Binding and Elution
16. 
 
Using clean forceps, add 2 DNA Capture Beads to each sample, which are now in Monarch 2 ml Tubes.
SECTION: Activity 2: HMW gDNA Binding and Elution
17. Add 550 µL isopropanol to each sample, close the cap, and invert slowly and gently 25–30 times. 

 

SECTION: Activity 2: HMW gDNA Binding and Elution
18. Remove and discard the excess liquid by pipetting. 
 

SECTION: Activity 2: HMW gDNA Binding and Elution
18.1. To remove the liquid, you can use either of the methods pictured below. In the past, the right method has yielded more liquid and interfered less with the beads.
 
Images from NEB Monarch protocol.
SECTION: Activity 2: HMW gDNA Binding and Elution
19. Add 500 µL  gDNA Wash Buffer, close the cap, and mix by inverting the tube 2–3 times. Then remove the gDNA Wash Buffer using the same method as the previous step. 
 
 


SECTION: Activity 2: HMW gDNA Binding and Elution
20. Repeat Step 4 and remove the gDNA Wash Buffer.


SECTION: Activity 2: HMW gDNA Binding and Elution
21. Place a labeled bead retainer into each Monarch Collection Tube II. Pour the beads from the 2 ml tube into the bead retainer and close the cap. You can discard the 2 ml tube. 
 

SECTION: Activity 2: HMW gDNA Binding and Elution
22. Use a mini centrifuge to remove any residual wash buffer from the beads. Only spin out for a moment (≤1 second) so as not to dislodge the gDNA from the beads.


SECTION: Activity 2: HMW gDNA Binding and Elution
23. Pull out the bead retainer from the first collection tube, pour the beads into the other labeled 2 ml tube, and insert the used bead retainer into the labeled 1.5 ml microfuge tube for later use during elution. Discard the used collection tube.

 
SECTION: Activity 2: HMW gDNA Binding and Elution
24. Immediately add 100 µL Elution Buffer II onto the glass beads and incubate for 10 min at 56 °C in a thermal mixer with agitation at   . 
 





SECTION: Activity 2: HMW gDNA Binding and Elution
24.1. At 3 minutes, check to make sure the beads are not stuck to the bottom of the tube by tilting the tube almost horizontally and gently shaking. If they become stuck for too long, the DNA will not release properly from the beads. 
 

SECTION: Activity 2: HMW gDNA Binding and Elution
24.2. Repeat the above process at 7 minutes.


SECTION: Activity 2: HMW gDNA Binding and Elution
25. After incubating, pour the eluate and beads into the bead retainer, which should already be inserted into a 1.5 ml microfuge tube. 
 

 

SECTION: Activity 2: HMW gDNA Binding and Elution
26. Centrifuge each sample for 30 s at 12000 x g  to separate the eluate from the glass beads. After separating, discard the beads and retainer. 



SECTION: Activity 2: HMW gDNA Binding and Elution
27. Pipette the eluate up and down 5–10 times with a wide bore pipette tip and ensure any visible DNA aggregates are dispersed. 
SECTION: Activity 2: HMW gDNA Binding and Elution
27.1. After pipetting, incubate at 37 °C  for 30-60 minutes,  at room temperature, or for > 1440 min   at 4 °C . Pipette up and down 5-10 times again before analyzing or using the HMW DNA.