TITLE: High-Molecular-Weight SPRI-aided DNA extraction from Mimulus (Phrymaceae) leaf tissue
AUTHORS: Bolívar Aponte Rolón

[DESCRIPTION]
This protocol is adapted from Russo et al. 2023 methods for high-molecular weight DNA extraction. I modified the protocol to use reagents and incubation conditions used by the Ferris Lab at Tulane University to extract DNA from Mimulus spp. (monkeyflower) tissue (leaf and buds), see protocol. The original CTAB:Chloroform protocol used by the Ferris lab traces back to the Willis Lab at Duke University. The objective of these DNA extractions are to collect fungal DNA present in leaf tissue. I have incorporated sample preparation procedures used by the Arnold Lab at The University of Arizona to eliminate contamination of samples and optimize DNA extractions from preserved leaf tissue for future fungal ITS Illumina MiSeq sequencing. Solid phase reversible immobilization beads (SPRI) utilized are adapted from Rohland and Reich 2012 and Liu et al 2023. All extractions were performed in a sterile biosafety cabinet to prevent sample contamination.

Prior to being placed in CTAB, photosynthetic tissue from plants was surface sterilized with sequential washes in 95% EtOH (10 s) , 0.5% sodium hypochlorite (1 min), and 70% EtOH (1min) and air dried under sterile conditions. Due to the small size of monkeyflower plants, the maximum amount of leaf tissue produced per host were placed in 750- 1000 µL of CTAB buffer and kept at RT until DNA extraction process.

*Several aspects of this protocol, mainly those with reagent amounts and molarities, have changed compared to the Russo et al 2023 protocol and the Willis Lab standard CTAB extraction protocol from ca. 2010.



[BEFORE_START]
Make sure to…
Turn on the water bath to 60°C and heating block to 50°C.
Get liquid nitrogen.  
Get sample tubes/plates from the -80°C or 20°C freezer.
Keep samples frozen! (On dry ice).

If and only if…

You have a fresh homemade CTAB buffer that does not have polyvinylpyrrolidoneand β-mercaptoethanol will you add
these reagents to stock buffer.

Instructions 

Right before use of CTAB buffer; prior to starting extraction, add polyvinylpyrrolidone and β-mercaptoethanol. 
Dissolve 1.1 – 2.2 grams of Polyvinyl-Pyrrolidone (PVP) per each 50-100ml CTAB (1.1 – 2.2% of the total buffer vol.).

Once these have been added the shelf life of the buffer is only 2-3 days.

If tissue is in homemade CTAB buffer and no freeze drying/lyophilization was performed (jumped directly to DNA extraction) you can add 2.25 µL of β-mercaptoethanol per 700 µLL CTAB, as well as 8 -16 µL of   (1.1-2.2 % solution) per 700 µL CTAB.

DO EXTRACTIONS IN THE BIOSAFETY CABINET (except for grinding, and spinning steps).




[GUIDELINES]
General Notes
DNA Extractions for Illumina Seq. are the most sensitive. If you need high quality low contamination DNA that is high molecular weight, make sure to use the gentler methods of mixing. Use wide bore pipet tips to help reduce shearing of DNA. Do not vortex any samples to mix, just invert or gently tap/flick tubes. To reduce contamination, be sure to check that all samples come out of the first chloroform step with a clear aqueous layer, not cloudy. Sodium chloride (5M NaCl) helps reduce polysaccharide contamination (compared to the Isopropanol precipitation without the added NaCl). High salt concentrations also aid in DNA precipitation, so you should get more DNA precipited and cleaned with chloroform wash. 

Sample Handling 
Gloves must be worn at all times. Change gloves frequently and decontaminate often with DNA Away.
Pipetting must be done extremely carefully to minimize the risk of aerosols that can easily cause contamination between samples.
Use only unopened, sterile, aerosol-resistant pipette tips (filter tips) to minimize contamination of the pipette shaft and your samples. Clean the body of the pipettes regularly with DNA Away.
Discard pipette tips after each use to avoid cross-contamination. Eject pipette tips carefully to prevent aerosol formation or other splashing.
Remember to always clean the hood and any equipment with DNA Away and decontaminate where possible with UV for 30 minutes prior to use to avoid cross-contamination.
The pipettes used need to be calibrated annually.
Be careful not to touch the inside lid of tubes as it will cross-contaminate samples.


[STEPS]
SECTION: Sample preparation
1. Clean the biosafety cabinet, pipettes, tip boxes and any other instrument to be used with 70% EtOH, 10 % Bleach and 95 % EtOH.  Surface sterilize all instruments in biosafety cabinet with UV light for 30 min. 
SECTION: Sample preparation
3. Quickly after placing tissue in tubes, place tube cap and close tightly.
 

SECTION: Sample preparation
4. Place tubes in the -20 °C or -80 °C freezer. Until ready for freeze drying/lyophilization step.


SECTION: Freeze drying/Lyophilization
5. Once all your samples are cold, turn on the refrigerator for the lyophilizer and place the glass plate on the front.
 

SECTION: Sample preparation
2.1. Weigh empty 2 mL tube to determine how much tissue you need to place in tube.  

SECTION: Sample preparation
2. Transfer 0.15-0.3 grams of leaf tissue to new 2 mL tube. Leaving remaining leaf tissue in original 15 mL vial tube.
 
  

SECTION: Sample preparation
2.2. Weigh 2 mL tube with leaf tissue to determine if it has the correct amount. 
 

SECTION: Freeze drying/Lyophilization
6. Once the lyophilizer temperature is at -40 °C, turn on the vacuum pump.
 

SECTION: Freeze drying/Lyophilization
7. When the pressure is at the appropriate level, release the pressure by turning the white port 180 degrees.         
 

SECTION: Freeze drying/Lyophilization
8. Lyophilize samples for a minimum of 2880 min.
 


SECTION: Freeze drying/Lyophilization
7.1. Quickly take frozen tubes out of the freezer and place inside the lyophilizer chamber. Close the lid and close the port. 
SECTION: Pulverization
9. In a sterile laminar flow hood, remove the parafilm or lid from the tubes (being careful not to touch the inside lip of the cap) and add beads for homogenization. For all plant samples except mosses, add the following: 
 

SECTION: Pulverization
10. Tubes with beads can be placed in a -20 °C  freezer until ready to finish pulverization steps.

SECTION: Pulverization
11. Dip tubes/96-well plates in liquid nitrogen.
 

SECTION: Pulverization
12. Shake at 1500 strokes/minute for 3 minutes in GenoGrinder 2010 or 25-30 Hz for 3 minutes if using TissueLyzer LT.


 

 


SECTION: Pulverization
13. Centrifuge plate fast and briefly at 4000 rpm when using 96-well plate centrifuge or 10.000 rpm when using Corning LSE centrifuge to get powder off lids.
 

SECTION: Pulverization
14. Store in -20 °C or -80 °C until DNA extraction steps.

SECTION: Pulverization
9.1. Add 100 μL (2 beads) of the autoclaved 3.2 mm stainless steel beads. 
 


SECTION: Pulverization
9.2. Add 2-3 of the autoclaved 2 mm zirconium oxide beads to each tube with a sterile scoop. 
SECTION: Pulverization
9.3. Add 100 μL of the autoclaved stainless steel bead blend, (0.9-2.0mm) using a sterile scoop. Close the lids securely.
 
SECTION: DNA Extraction
15. Surface sterilize all workbench surfaces and instruments with a 10% solution of 0.5% sodium hypochlorite, 70% EtOH and 95% EtOH prior to placement in biosafety cabinet. Surface sterilize all
instruments in biosafety cabinet with UV light for 30 min. 

SECTION: DNA Extraction
16. Make Extraction Master Mix (Pre-warm to 60°C):
Measure out700 µL of CTAB extraction buffer per sample into 15-50 mL conical centrifuge tube. Add2.25 µL β-mercaptoethanol per 700 µL of CTAB extraction buffer to the same conical tube (0.3% of the total extraction buffer volume).

 Basic recipe:                                              
[(# of samples x 1.1) x 700 µL of CTAB] + [(# of samples + 1) x 2.25 µL β-mercaptoethanol]                             
 

  
 
 
SECTION: DNA Extraction
19. After 20 minutes incubation in step 18, add 4 µL of RNase A. 
  

SECTION: DNA Extraction
17. Add 700 µL of preheated Extraction Master Mix to each tube, replace the lids (if using strip tubes).
 

 


SECTION: DNA Extraction
18. Incubate tubes for 20 min in a 60 °C water bath. 

 

SECTION: DNA Extraction
20. Incubate at 60 °Cfor 10 min.
 

SECTION: DNA Extraction
21. All 200 µL of 5M NaCl (or 5M Sodium/Potassium acetate). To fully precipitate proteins and  polysaccharides in extraction buffer.
 
  


SECTION: DNA Extraction
22. Add 900 µL of chloroform/isoamyl (24:1) (1 volume) to each tube.

SECTION: DNA Extraction
23.  Place tubes in a nutating mixer for 10 min at 24 rpm.

 

 
     


SECTION: DNA Extraction
24. Centrifuge for 10 min at 4000rpm when 96-well plate centrifuge or 10 min at10.000 rpm when using Corning LSE centrifuge. 

 



SECTION: DNA Extraction
25. Carefully pipette off ~800 µL-900 µL of the top aqueous layer using filter pipet tips and
transfer to sterile new tubes or 96-well plate. 
 

  

SECTION: DNA Extraction
26. Repeat steps 22-24   .
 


SECTION: DNA Extraction
27. After repeating steps 22-24. Carefully pipette ~700 µL-800 µL of the top aqueous layer using wide-bore filter pipet tips and transfer to sterile new tubes or 96-well plate.
 

  
 


SECTION: SPRI aided isolation and purification of DNA
28.       Turn ON heating block to 50 °C and place 10 mM Tris-HCl or 1X TE buffer to pre-heat. 
    
SECTION: SPRI aided isolation and purification of DNA
29.   Add SPRI bead mixture according to the following:
  Volume of SPRI beads to add = 1.2 × DNA volume (from step 27)
 

 

  
  
   

SECTION: SPRI aided isolation and purification of DNA
30. Incubate for 30 min in a nutating mixer at 24 rpms (default).
SECTION: SPRI aided isolation and purification of DNA
31. Spin down for 5 s on centrifuge. 

SECTION: SPRI aided isolation and purification of DNA
32. Place the tubes on a magnetic rack for 5 min or wait until the
solution is clear.
 


SECTION: SPRI aided isolation and purification of DNA
33. Aspirate the cleared solution from the tubes, inspect pipette tips for bead residue, and discard.

 

SECTION: SPRI aided isolation and purification of DNA
34. Dispense 1 mL of fresh 80% ethanol to each tube and invert 25 times to resuspend bead pellet.
 
  


SECTION: SPRI aided isolation and purification of DNA
35. Spin down for 1-2 s on centrifuge. 
SECTION: SPRI aided isolation and purification of DNA
36. Place tubes on magnetic rack.
SECTION: SPRI aided isolation and purification of DNA
37. Aspirate out the ethanol, inspect pipette tips for bead residue, and discard.
SECTION: SPRI aided isolation and purification of DNA
38. Repeat for a total of three washes.   

SECTION: SPRI aided isolation and purification of DNA
39. Let beads air-dry for 1 min after final wash.

SECTION: SPRI aided isolation and purification of DNA
40. Take tubes out of the magnetic rack, add 50 μL of elution buffer (10 mM TRIS-HCl pH 8.0,
or 1X TE buffer) pre-heated to50 °C to each tube and pipette mix 5 times.
    

SECTION: SPRI aided isolation and purification of DNA
42. Incubate tubes at 37 °C for 15 min in benchtop shaking incubator at 20 rpm. 

 

SECTION: SPRI aided isolation and purification of DNA
41. Spin down for 1-2 s on centrifuge. 
SECTION: SPRI aided isolation and purification of DNA
43. Place tubes in magnetic rack and let sit for 15 min-30 min. 
 

SECTION: SPRI aided isolation and purification of DNA
44.  Collect ~50 µL of supernatant and transfer to a new tube.

SECTION: SPRI aided isolation and purification of DNA
45. Place in tube stand and let sit at RT overnight. 
 
    
SECTION: SPRI aided isolation and purification of DNA
46. Store DNA at -20 °C or -80 °C until ready for quantification with Qubit or PicoGreen assay and downdtreams analyses.