Jan 09, 2023
Wisecaver Lab CTAB-based extraction of high molecular weight DNA from photosynthetic sea slugs
- Jennifer H Wisecaver1,
- Raeya Ogas1,
- Pax Tomko1,
- Gregory Gavelis2
- 1Purdue University;
- 2Purdue University, Bigelow Laboratory for Ocean Sciences
Protocol Citation: Jennifer H Wisecaver, Raeya Ogas, Pax Tomko, Gregory Gavelis 2023. Wisecaver Lab CTAB-based extraction of high molecular weight DNA from photosynthetic sea slugs . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxp81zl8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 15, 2021
Last Modified: January 09, 2023
Protocol Integer ID: 51580
Abstract
Protocol for CTAB-based extraction of high molecular weight DNA from photosynthetic sea slugs. This protocol is optimized for samples with high mucopolysaccharide contents and is derived from:
Extraction of high molecular weight DNA from molluscs. 1993. Trends in Genetics.Vol 9: pg 407
Materials
Required sterile tools and disposables:
- 5 mL and 1.5 ml LoBind microcentrifuge tubes
- Mortar & Pestle
- Scalpel, if performing slug dissection
- Magnetic stand for Ampure beads
- Wide-bore pipette tips
- Water bath
Other required solutions/chemicals:
- TrisHCL pH 8
- NaCL
- Beta-mercaptoethanol
- EDTA
- proteinase K
- Chloroform:Isoamyl alcohol (24:1)
- Phenol:Chloroform:Isoamyl alcohol (25:24:21)
- Isopropanol
- 100% Ethanol
- RNaseA (100 mg/mL)
- TE buffer
- Ampure Beads
Safety warnings
At steps involving beta-mercaptoethanol and chloroform (**carcinogens**) should be performed in the chemical fume hood.
Prepare CTAB DNA extraction buffer
Prepare CTAB DNA extraction buffer
Prepare 100 mL CTAB buffer:
- 100 mL Ultrapure distilled water
- 1.576 g TrisHCL ph 8 (100 mM)
- 0.584 g EDTA (20 mM)
- 8.82 g NaCl (1.4 M)
- 10 mg Proteinase K (0.1 mg/mL)
Note: This stock buffer is sufficient for 9-10 extractions.
On the day of an extraction, per sample: add 20 µL beta-mercaptoethanol (BME) to 10 mL CTAB buffer.
Safety information
BME is a carcinogen.
ALL steps containing BME should be performed in the chemical fume hood.
All BME liquid waste should be ejected or decanted into the amber bottle labeled 'BME Waste' inside the fume hood. Any plasticware that comes into contact with BME needs to be properly disposed of in the sharps bin inside the fume hood labeled 'Mutagen/Carcinogen Sharps Do Not Autoclave'.
Use a water bath to preheat CTAB+BME buffer at 60 °C in a water bath.
Prepare equipment and reagents
Prepare equipment and reagents
On the day of the of extraction:
- Chill centrifuge to 4 °C
- Get liquid nitrogen
- Chill mortar and pestle in the -20 °C freezer
- Bring Ampure beads to room temperature (~ 20 °C )
- Prepare 5 mL of 70% Ethanol
Decide if performing DNA extraction using whole slugs or dissected tissue.
Dissection of brain tissue31 steps
Dissections must be performed using living slugs (i.e., not slugs that were previously frozen). Euthanize slugs by cutting of their head. Dissect the head and remove the brain (light colored tissue that does not contain any plastids). This minimizes contamination by gut contents.
Use mortar + pestle + liquid nitrogen to grind the sample(s), either whole/partial slug or dissected brain tissue) to a powder
Remove mucopolysaccharides
Remove mucopolysaccharides
Transfer pulverized sample to a 50 mL centrifuge tube with 10 mL preheated CTAB+BME buffer
Incubate at 60 °C for 00:30:00
30m
Add 10 mL of chloroform:isoamyalcohol. Mix well.
Safety information
Chloroform is a carcinogen.
ALL steps containing Chloroform should be performed in the chemical fume hood.
All Chloroform liquid waste should be ejected or decanted into the amber bottle labeled 'Phenol:Chloroform Waste' inside the fume hood. Any plasticware that comes into contact with Chloroform needs to be properly disposed of in the sharps bin inside the fume hood labeled 'Mutagen/Carcinogen Sharps Do Not Autoclave'.
Centrifuge at 7700 x g for 00:10:00
10m
Transfer 2.5 mL aqueous phase to a 5 mL lo-bind microcentrifuge tube.
Precipitate nucleic acids
Precipitate nucleic acids
5m
5m
Add 2/3 volume (1.7 mL ) isopropanol and gently invert tube for 00:05:00 at room temperature.
5m
Centrifuge for 0 mL at 10000 x g . Discard th supernatant.
Add 300 µL of 70% ethanol to the DNA pellet. Use a pipette tip to dislodge the pellet from the wall of the microcentrifuge tube.
Centrifuge for 00:05:00 at 10000 x g . Discard the supernatant.
5m
Use a pipette to remove the remainder of teh supernatant from the tube. Be careful not to disrupt the pellet.
Gently lay the tube on its side on a sterile work surface with the cap open. Allow the tube to air dry in this position for 00:10:00 .
10m
Degrade RNA and extract DNA
Degrade RNA and extract DNA
Resuspend the pellet in 120 µL of TE buffer.
Add DNAase-free RNase A to a final concentration of 1 ug/mL. Incubate at 37 °C for 00:30:00 .
30m
Add 120 µL phenol:chloroform:isoamylalcohol.
Safety information
Chloroform is a carcinogen.
ALL steps containing Chloroform should be performed in the chemical fume hood.
All Chloroform liquid waste should be ejected or decanted into the amber bottle labeled 'Phenol:Chloroform Waste' inside the fume hood. Any plasticware that comes into contact with Chloroform needs to be properly disposed of in the sharps bin inside the fume hood labeled 'Mutagen/Carcinogen Sharps Do Not Autoclave'.
Centrifuge for 00:10:00 at 10000 x g . Transfer 60 µL of the aqueous phase to a clean 1.5 mL lo-bind microcentrifuge tube. Discard the remainder.
Safety information
This is the last supernatant and plastic waste that needs to be disposed of in the phenol:chloroform or BME waste containers. All waste in the remaining steps can go in the standard lab 'look-alike' waste bin.
10m
Bead-purify DNA
Bead-purify DNA
Resuspend the ampure beads by quickly vortexing. Add 108 µL ampure bead solution to the aqueous phase sample. Mix gently by pipetting up and down 10 times using a wide-bore pipette tip.
Incubate at room temperature (20 °C ) for 00:05:00 .
5m
Place tube in the magnetic stand until the solution becomes clear (approx. 00:02:00 ).
2m
With the tube still in the magnetic stand, carefully discard the clear supernatant
Keeping the tube in the magnetic stand, add 200 µL of 70% ethanol. Incubate for 00:00:30 . Gently remove the clear supernatant, being careful not to disturb the beads.
Repeat this wash step three times.
30s
Ensure that the ethanol is completely removed and leave the tube top open to dry for 00:05:00 . Do not 'overdry' (i.e., to the point that the bead streak is cracking)
5m
Remove the tube from the magnetic stand. Add 60 µL TE to dissolve the DNA.
Mix gently by pipetting up and down 10 times using a wide-bore pipette tip.
Incubate at room temperature (20 °C ) for 00:05:00
5m
Place tube in the magnetic stand until the solution becomes clear (approx. 00:01:00 ).
1m
Transfer the clear DNA suspension to a new tube. Discard the old tube with the beads.
QC and cleanup
QC and cleanup
Make sure all chloroform waste is properly disposed of in the chemical fume hood.
Wipe down the chemical fume hood and the benchtop with 70-75% ethanol.
Assess DNA integrity using the TapeStation. Make sure you reserve time on the TapeStation Teams calendar.
While the TapeStation is running, measure the DNA concentration using the Qubit.
If the TapeStation reports a high DNA integrity (DIN) score (≥ 7) and DNA concentration ≥ 20 ng/uL, then you have successfully isolated DNA for whole genome sequencing!
Cap your sample tube tightly. Make sure each tube is labeled on the cap and vertically on the side with the sample's unique identifier*. Wrap the cap with parafilm and place sample in the 'Novogene' box in the full sized -20 freezer.
*The identifier should start with a letter and be no more than 12 characters long. Write it carefully using a fine tip permanent marker as this tube will be shipped off for DNA sequencing.