TITLE: High Molecular Weight Total DNA Extraction from plant tissues for Long Read Sequencing
AUTHORS: Subash  Rai

[DESCRIPTION]
This protocol was developed as a research within GIH collaborative projects for a sample where a species of Neptunia leaves sample was problematic in our previously developed protocol. At step 21 of previous protocol, the solution forms either a brownish mark on the top layer of the solution or the whole solution depending on the starting sample amount which resulted brown CTAB-DNA complex. In this protocol, the problematic steps for a particular sample is improved to extract high quality High Molecular Weight (HMW) DNA >60kb. The DNA quality was assessed in Qubit, NanoDrop, TapeStation, and Oxford Nanopore Technologies. Using a LSK109 ligation chemistry and R9.4 flow cell, a total yield of 24gb with N50 29kb was generated in MinION sequencing platform.  

[BEFORE_START]
Prepare the following buffers and solutions before starting the experiment:

Lysis Buffer 
      Tris-HCl  100mM    EDTA  20mM    CTAB (w/v)  4%    NaCl  1.4M    PVP 360k (w/v)  1%    β-mercaptoethanol (add just before use)]  2%  
Combine the reagents given in the table below. 
      1M Tris-HCl (pH = 8)  5ml    0.5M EDTA (pH = 8)  2ml    CTAB powder  2g    PVP  0.5g    NaCl  4g  
Adjust the final volume to 50 mL with Nuclease free water/lab grade water. Store at -80 Room temperature for up to 3-4months. 

High-salt TE buffer 
      EDTA  2mM    Tris-HCl  10mM    NaCl  1M   
 Combine the reagents given in the table below 
      NaCl  581 mg    0.5M EDTA (pH=8)  40 μl    1M Tris-HCl (pH=8)  100 μl  
 Complete to 10 mL with Distilled water Ultra-Pure. Autoclave it for long-term (1 year) storage.

Binding buffer (20% PEG8000 and 3M NaCl):
Add 2 g PEG 8000 and 1.75 g NaCl in 10 mL nuclease free water and mix well until it turns as a clear solution and store at cold room or 4-7°C.

Beads solution: 
      Dynabeads™ M-270   Carboxylic Acid  4%    PEG8000  18%    NaCl  1M    Tris-HCl pH-8  10mM    EDTA pH-8  1mM  
First prepare the required volume of the solution except Dynabeads.
Keep the Dynabeads at RT for at least 15 min. Mix well by vortexing, then take 4% of the beads solution (v/v) immediately.
Wash the beads with nuclease free water 3 times. Resuspend the beads pellet completely while washing.
Add the beads solution and store the beads solution at 4 °C.
Keep the beads solution at 4 Room temperature for at least 15 min and mix well before using it. 

[GUIDELINES]
Starting materials:
Young and healthy tissues are ideal samples for HMW DNA extraction. The amount of sample required depends on the plant genome size. More material is required for small genome plants when compared to bigger genome plants (of equivalent sample quality).

Handling of HMW DNA:

Always use wide-bore pipette tips as recommended in the protocol.
Allow the DNA to stand in elution buffer   at Room temperature or tap the tube gently. 
 

Avoid repeated cycle of freezing and thawing. Aliquot the required amount of DNA in multiple tubes before storing at -20 °C/ -80 °C.

Citations
 

 


[STEPS]
SECTION: Tissues preparation and lysis
1. Take 10 mL lysis buffer and warm it at 60 °C for 15-20 min.

SECTION: Tissues preparation and lysis
2. Take ~1 L of liquid nitrogen (LN2) in Dewar Flask that requires for chilling mortar and pestle and grinding the tissues.

SECTION: Tissues preparation and lysis
3. Take dry ice in an esky/insulated container for later steps.
SECTION: Tissues preparation and lysis
4. Grind 500 mg to 1000 mg healthy young fresh/snap frozen/frozen tissues in mortar and pestle chilled with LN2 to fine powder. 
 

SECTION: Tissues preparation and lysis
5. Keep a 15 ml falcon tube on the dry ice for 5 min then swirl the ground powder with LN2 and pour directly into the falcon tube while keeping the falcon tube on the dry ice.

SECTION: Tissues preparation and lysis
6. Keep the lid half-opened and let LN2 to evaporate.
SECTION: Tissues preparation and lysis
7. Take out the tube and add 10 mL prewarmed lysis buffer (at 60 °C) with freshly added 200 µL β- mercaptoethanol.

SECTION: Tissues preparation and lysis
8. Mix well by inverting the tubes (~100 times) until the solution become more homogenous. In some sample, solution may not be homogenous but form whiteish clumps (it is normal) and incubate at 60 °C in thermomixer at 300 rpm for 30 min.

SECTION: Tissues preparation and lysis
9. Add 200 µL Proteinase K (stock conc=20 mg/mL) after 15 min of incubation.

SECTION: Tissues preparation and lysis
10. Mix well by inverting the tube (15-20 times) and continue the incubation.
SECTION: Tissues preparation and lysis
11. Spin the solution at 3000 x g, 5 min. 
 

SECTION: Tissues preparation and lysis
12. Take an equal volume of the supernatant in two fresh 15 ml falcon tubes using P1000 wide bore pipette tips.
SECTION: Extraction of raw HMW DNA
13. Add an equal volume of Chloroform:Isoamyl alcohol (24:1) into the solution.
SECTION: Extraction of raw HMW DNA
14. Mix the solution by inverting the tube until a milky colour appears (~100 times) and centrifuge at 3000 x g, 10 min. 

SECTION: Extraction of raw HMW DNA
15. Transfer the aqueous phase to a new 15ml falcon tube without disturbing interface layer.
SECTION: Extraction of raw HMW DNA
16. Add an equal volume of Chloroform:Isoamyl alcohol (24:1) into the solution.
SECTION: Extraction of raw HMW DNA
17. Mix the solution by inverting the tubes ~100 times and centrifuge at 3000 x g, 10 min.

SECTION: Extraction of raw HMW DNA
18. Transfer 1 ml aqueous phase to 2 ml LoBind tube without disturbing the interface layer (much thinner than the first extraction). It requires multiple 2 ml LoBind tube.
 

SECTION: Extraction of raw HMW DNA
19. Add half volume of Ammonium acetate (7.5 M). Mix well by inverting the tubes and incubate for 10 min at 60 Room temperature
SECTION: Extraction of raw HMW DNA
20. Centrifuge at 13000 x g, 10 min .
SECTION: Extraction of raw HMW DNA
22. Resuspend the pellet with 1 mL 70% ethanol (freshly prepared) using a wide bore P1000 pipette tip and transfer all into a 2ml LoBind DNA tube.
SECTION: Extraction of raw HMW DNA
23. Rinse the tube with additional 1 mL 70% ethanol to collect remaining CTAB-DNA complex. Perform the same for another 15 ml falcon tube.

SECTION: Extraction of raw HMW DNA
24. Incubate 2 ml tubes for 5 min at 60 Room temperature in a HulaMixer at 9 rpm.

SECTION: Extraction of raw HMW DNA
25. Spin the tube at 13000 x g, 5 min and discard the supernatant.

SECTION: Extraction of raw HMW DNA
26. Repeat washing steps    &   once with 2 mL 70% ethanol.

SECTION: Extraction of raw HMW DNA
27. Keep the tubes under fume hood for 5 min to remove any traces of ethanol.

SECTION: Extraction of raw HMW DNA
28. Resuspend the DNA pellet in 200 µL of prewarmed (60 °C) High-salt TE buffer.

SECTION: Extraction of raw HMW DNA
29. Add 4 µL RNaseA and incubate at 37 °C for 15-20 min.

SECTION: Beads Purification of HMW DNA
30. Add 100 µL ammonium acetate (7.5 Molarity (M)) mix well and incubate it for 10 min at 37 Room temperature. Shake it once every 5 min.
SECTION: Beads Purification of HMW DNA
31. Spin the tube at 13000 x g, 3 min and transfer the supernatant using wide-bore pipette tips into a fresh tube.

SECTION: Beads Purification of HMW DNA
32. Add equal volume of binding buffer and mix well by inverting the tube.
SECTION: Beads Purification of HMW DNA
33. Add 150 µL beads solution (8-9 million beads) and incubate it for 30 min at 37 Room temperature in a HulaMixture.

SECTION: Beads Purification of HMW DNA
34. Place the tube in magnetic rack for 2-3 min and remove the supernatant and wash the beads with 500 µL freshly prepared 70% ethanol (clumping of beads may appear but try to dislodge by inverting the tube several times).

SECTION: Beads Purification of HMW DNA
35. Wash the pellet once again with 500 µL freshly prepared 70% ethanol.

SECTION: Beads Purification of HMW DNA
36. Add 75 µL prewarmed (at 50 °C) 10 millimolar (mM) Tris-HCl (elution buffer) pH 8 and incubate at 37 Room temperature for 15 min.

SECTION: Beads Purification of HMW DNA
37. Place the tube back in the magnetic rack and leave it for 5 min.

SECTION: Beads Purification of HMW DNA
38. Remove the supernatant in the fresh 1.5ml LoBind DNA tube. 
 

SECTION: Beads Purification of HMW DNA
35.1. Take out the tubes from magnetic rack and add 500 µL freshly prepared 70% ethanol.
SECTION: Beads Purification of HMW DNA
35.2. Dislodge the beads by inverting the tubes.
SECTION: Beads Purification of HMW DNA
35.3. Place the tubes back to the magnetic rack for 2 min and remove the supernatant.
SECTION: Beads Purification of HMW DNA
39. Assess DNA quality in NanoDrop, Qubit, and TapeStation/PFGE 
SECTION: Extraction of raw HMW DNA
21. Transfer the supernatant in a fresh 2ml LoBind tube and add equal volume of Isopropanol (>98%), mix well, and incubate  for 10 min  at Room temperature .

SECTION: Worked Results
40. 



General Quality check:
  
 
QC check in Oxford Nanopore Sequencing: