High-Molecular-Weight SPRI-aided DNA extraction from Mimulus (Phrymaceae) leaf tissue
Bolívar Aponte Rolón
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Abstract
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 polyvinylpyrrolidone and 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
2.25µLof β-mercaptoethanol per700µLL 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).
Steps
Sample preparation
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 0h 30m 0s.
Transfer 0.15-0.3 grams of leaf tissue to new 2 mL tube. Leaving remaining leaf tissue in original 15 mL vial tube.
Weigh empty 2 mL tube to determine how much tissue you need to place in tube.
Weigh 2 mL tube with leaf tissue to determine if it has the correct amount.
Quickly after placing tissue in tubes, place tube cap and close tightly.
Place tubes in the -20°C or -80°C freezer. Until ready for freeze drying/lyophilization step.
Freeze drying/Lyophilization
Once all your samples are cold, turn on the refrigerator for the lyophilizer and place the glass plate on the front.
Once the lyophilizer temperature is at -40°C, turn on the vacuum pump.
When the pressure is at the appropriate level, release the pressure by turning the white port 180 degrees.
Quickly take frozen tubes out of the freezer and place inside the lyophilizer chamber. Close the lid and close the port.
Lyophilize samples for a minimum of 48h 0m 0s.
Pulverization
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:
Add 100 μL (2 beads) of the autoclaved 3.2 mm stainless steel beads.
Add 2-3 of the autoclaved 2 mm zirconium oxide beads to each tube with a sterile scoop.
Add 100 μL of the autoclaved stainless steel bead blend, (0.9-2.0mm) using a sterile scoop. Close the lids securely.
Tubes with beads can be placed in a -20°C freezer until ready to finish pulverization steps.
Dip tubes/96-well plates in liquid nitrogen.
Shake at 1500 strokes/minute for 3 minutes in GenoGrinder 2010 or 25-30 Hz for 3 minutes if using TissueLyzer LT.
Equipment
| Value | Label |
|---|---|
| Tissuelyser LT | NAME |
| Qiagen | BRAND |
| 85600 | SKU |
| Tissue and cell lyser | SPECIFICATIONS |
Centrifuge plate fast and briefly at 4000rpm,0h 0m 0s when using 96-well plate centrifuge or 10.000rpm,0h 0m 0s when using Corning LSE centrifuge to get powder off lids.
Store in -20°C or -80°C until DNA extraction steps.
DNA Extraction
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 for0h 30m 0s .
Make Extraction Master Mix (Pre-warm to 60 ° C):
Measure ou700µL of CTAB extraction buffer per sample into 15-50 mL conical centrifuge tube. Ad2.25µL β-mercaptoethanol per700µL 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 0 µL of CTAB] + [(# of samples + 1) x 2.25 5 µL β-mercaptoethanol]
Add 700µL of preheated Extraction Master Mix to each tube, replace the lids (if using strip tubes).
Incubate tubes for 0h 20m 0s in a 60°C water bath.
After 20 minutes incubation in step 18, add of RNase A 4µL of RNase A.
Incubate at 60°Cfor 0h 10m 0s.
All 200µL of 5M NaCl (or 5M Sodium/Potassium acetate). To fully precipitate proteins and polysaccharides in extraction buffer.
Add 900µL of chloroform/isoamyl (24:1) (1 volume) to each tube.
Place tubes in a nutating mixer for 0h 10m 0s at 24 rpm.
Equipment
| Value | Label |
|---|---|
| S0500 Mini Nutating Mixer | NAME |
| VWR | BRAND |
| 82007-202 | SKU |
| Mixer | SPECIFICATIONS |
Centrifuge for 0h 10m 0s at 4000rpm when 96-well plate centrifuge or 0h 10m 0s at10.000rpm,0h 0m 0s when using Corning LSE centrifuge.
Carefully pipette off ~ - of the top aqueous layer 800µL-900µL of the top aqueous layer using filter pipet tips and
transfer to sterile new tubes or 96-well plate.
Repeat steps 22-24 .
After repeating steps 22-24. Carefully pipette ~700µL-800µL of the top aqueous layer using wide-bore filter pipet tips and transfer to sterile new tubes or 96-well plate.
SPRI aided isolation and purification of DNA
Turn ON heating block to `50°C` and place 10 mM Tris-HCl or 1X TE buffer to pre-heat.
Add SPRI bead mixture according to the following:
Incubate for 0h 30m 0s in a nutating mixer at 24 rpms (default).
Spin down for 0h 0m 5s on centrifuge.
Place the tubes on a magnetic rack for 0h 5m 0s or wait until the
solution is clear.
Aspirate the cleared solution from the tubes, inspect pipette tips for bead residue, and discard.
Dispense 1mL of fresh 80% ethanol to each tube and invert 25 times to resuspend bead pellet.
Spin down for 1-2 s on centrifuge.
Place tubes on magnetic rack.
Aspirate out the ethanol, inspect pipette tips for bead residue, and discard.
Repeat for a total of three washes.
Let beads air-dry for 0h 1m 0s after final wash.
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 t50°C to each tube and pipette mix 5 times.
Spin down for 1-2 s on centrifuge.
Incubate tubes at for 37°C for 0h 15m 0s in benchtop shaking incubator at 20 rpm.
Place tubes in magnetic rack and let sit for - . 0h 15m 0s-0h 30m 0s.
Collect ~50µL of supernatant and transfer to a new tube.
Place in tube stand and let sit at RT overnight .
Store DNA at -20°C or -80°C until ready for quantification with Qubit or PicoGreen assay and downdtreams analyses.
