protocols.io | https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1 August 9, 2023 1/8
Aug 09, 2023
High molecular weight DNA extraction for marine macroalgal
tissue
DOI
dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1
Malia Moore , Taylor S. Steele
Scripps Institution of Oceanography
Malia Moore
Scripps Institution of Oceanography, Salk Institute for Biol...
1 1
1
DOI: dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1
Protocol Citation: Malia Moore, Taylor S. Steele 2023. High molecular weight DNA extraction for marine macroalgal tissue.
protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/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: May 08, 2023
Last Modified: August 09, 2023
Protocol Integer ID: 81551
Keywords: Lyophilizing algal tissue, DNA extraction, Lysis and first precipitation, Final precipitation, Column cleanup
protocols.io | https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1 August 9, 2023 2/8
Abstract
This protocol details high molecular weight DNA extraction for marine macroalgal tissue. Marine macroalgae contain a
variety of unique cell wall components including sulfated polysaccharides and polyphenolics. These components often
co-elute with high molecular weight (HMW) DNA and lead to reduced library prep and sequencing outcomes. This protocol
incorporates polyvinylpolypyrrolidone (PVPP) and β-mercaptoethanol (BME) to reduce polyphenolic contamination, and an
early salting out step with potassium acetate (KOAc) to address polysaccharides. This protocol is largely adapted from an
Oxford Nanopore HMW DNA extraction from Arabidopsis leaves, which incorporates the QIAGEN Blood and Cell Culture
DNA Midi Kit for column cleanup. The DNA product often requires additional cleanup after elution, and we suggest the
BluePippin 15kb size selection for all HMW applications.
Attachments
711-1533.pdf
55KB
Guidelines
Marine macroalgae contain a variety of unique cell wall components including sulfated polysaccharides and
polyphenolics. These components often co-elute with high molecular weight (HMW) DNA and lead to reduced library prep
and sequencing outcomes. This protocol incorporates polyvinylpolypyrrolidone (PVPP) and β-mercaptoethanol (BME) to
reduce polyphenolic contamination, and an early salting out step with potassium acetate (KOAc) to address
polysaccharides.
1
This protocol is largely adapted from an Oxford Nanopore HMW DNA extraction from
Arabidopsis
leaves, which incorporates the QIAGEN Blood and Cell Culture DNA Midi Kit for column cleanup.
2
The DNA product often
requires additional cleanup after elution, and we suggest the BluePippin 15kb size selection for all HMW applications.
Additional tips:
In the eld or in lab, it is vital to scrape off all surface epiphytes and wash the sample in clean water before ash
freezing to reduce contaminants common in the marine environment that confound genome assembly.
Marine macroalgae are incredibly diverse in biochemical content, so individual seaweeds may require troubleshooting.
Suggested alterations include varying input tissue type or quantity, increasing CTAB or BME percent, or adding a
second chloroform separation.
It may be necessary to carry out extractions of the same tissue in parallel to yield sucient DNA, especially when large
losses from BluePippin are expected. It is not suggested to combine multiple extractions onto the same column, as
this may lead to overloading and a dirty sample. This protocol as written, paired with BluePippin, has produced
sequencing-quality DNA for Nanopore from a red alga
Porteria hornemanii
and a brown alga
Macrocystis pyrifera
. For
P. hornemanii
, a single
20 mL
extraction produced sucient DNA for sequencing, but for
M. pyrifera
, three parallel
extractions of
20 mL
were necessary.
protocols.io | https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1 August 9, 2023 3/8
Materials
Equipment:
Lyophilizer
Stir plate
Heat block or water bath
Vortex
Mortar and pestle
Refrigerated centrifuge for spins up to 3,500 xg with
50 mL
Suggested: Sage Science BluePippin
Consumables:
Stock solution:
1 Molarity (M)
Tris-HCl,
9.5
Stock solution:
5 Molarity (M)
sodium chloride (NaCl)
Stock solution:
500 millimolar (mM)
ethylenediaminetetraacetic acid (EDTA)
Stock solution:
5 Molarity (M)
potassium acetate (KOAc)
Cetyltrimethylammonium bromide (CTAB)
Polyethylene glycol (PEG) 8000
β-mercaptoethanol (BME)
Polyvinylpolypyrrolidone (PVPP)
RNase A,
100 mg/mL
(eg. QIAGEN Mat. #1007885)
100% isopropanol
95-100% ethanol
Nuclease-free water
Blood & Cell Culture DNA Mini Kit (25) Qiagen Catalog #13323
Tris-EDTA (TE) buffer
50 mL
Falcon Tubes
DNA LoBind Tube 1.5ml Eppendorf Catalog #022431021
Suggested: Sage Science High Pass Plus Cassette (BPLUS10 or BPLUS03) for BluePippin
protocols.io | https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1 August 9, 2023 4/8
1
Flash-freeze algal tissue in liquid nitrogen (target ≥
5 g
wet tissue).
2 Quickly transfer sample to lyophilization container and freeze dry for 36-48 hours.
3 Macerate the tissue with a clean spatula to increase surface area and put on the lyophilizer for
another
24:00:00
.
4
Remove and refrigerate with desiccant for immediate use, or store at
-80 °C
for longer
periods.
5
Prepare desired volume of Carlson lysis buffer (
100 millimolar (mM)
Tris-HCl,
9.5
,
2% CTAB,
1.4 Molarity (M)
NaCl, 1% PEG 8000,
20 millimolar (mM)
EDTA) and mix
Overnight
on a magnetic stirrer. The stock solutions suggested under consumables will
yield a homogenous buffer with no precipitate.
6
Pre-heat a heat block or water bath to
65 °C
and place in a fume hood.
7
For each extraction, transfer
20 mL
of Carlson lysis buffer to a 50-ml Falcon tube.
8
In a fume hood, add
400 µL
BME (originally
50 µL
) and mix by vortexing. Pre-warm
the solution to
65 °C
for
00:30:00
before starting the extraction.
9
Scoop 0.5 teaspoons lyophilized plant tissue into a clean mortar and add
50-100 mg
powdered PVPP. Grind with pestle for ~
00:00:30
, until tissue is powdered and combined,
but not long enough to introduce signicant moisture. Move immediately into DNA extraction.
1d
1d
30m
30s
Lyophilizing algal tissue
Setting up the DNA extraction
Lysis and first precipitation
protocols.io | https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1 August 9, 2023 5/8
10 Pour tissue into the warm buffer. Invert 5 times.
11
Add
40 µL
of RNase A and vortex for
00:00:05
.
12
Optional: If using a heat block with mixing, set the block (still at
65 °C
) to mixing at
300 rpm, 00:05:00
.
13
Incubate for
01:00:00
at
65 °C
.
13.1 Invert 10 times every 15 minutes.
13.2
At 30 minutes, add another
40 µL
of RNase A, inverting 10 times to combine.
14
Allow the tubes to cool down to
Room temperature
for
00:10:00
.
15
Add
20 mL
chloroform and vortex for two pulses of
00:00:05
each.
16
Centrifuge the tubes at
3500 x g, 4°C, 00:15:00
.
17 In a fume hood, transfer the top layer of lysate from each tube to a new 50-ml Falcon tube,
without disturbing the interphase.
Note
Tip: The lysate layer should be
14-18 mL
of solution, but it is recommended to use
widebore tips, transferring
1 mL
at a time. Tips can also be widened by cutting
standard P1000 tips.
5s
1h
10m
5s
15m
protocols.io | https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1 August 9, 2023 6/8
18
Mix supernatant with 0.4X
5 Molarity (M)
potassium acetate (KOAc) at
Room temperature
, inverting at least 10 times to combine, then incubate
On ice
for
00:20:00
.
19
Centrifuge the tubes at
3500 x g, 4°C, 00:45:00
.
20 Remove and retain the supernatant.
Note
Tip: This may best be done by pouring slowly and observing the polysaccharide-salt pellet,
which may be mobile. Leave some liquid behind in favor of avoiding the pellet.
21
Add 0.7X volumes of isopropanol. Invert 10 times. Incubate at
-80 °C
for
00:15:00
.
Note
Do not extend this incubation.
22
Centrifuge the sample at
3500 x g, 4°C, 00:45:00
.
Note
Tip: If available, a xed-angle centrifuge will create a pellet on the wall of the tube that has
greater surface area for dissolution in step 24 (as compared to a conical pellet at the base
of a falcon tube from a swinging bucket).
23 Discard the supernatant without disturbing the pellet. Use sterile wipes to absorb the liquid on
the tube walls, being careful not to disturb the pellet.
24
To each pellet, add
10 mL
G2 buffer, from the QIAGEN kit. Incubate at
50 °C
for 30-
60 minutes, or until the pellet is dissolved. Swirl the pellet to mix but do not try to pipette or
vortex.
20m
45m
15m
45m
protocols.io | https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1 August 9, 2023 7/8
25
Equilibrate a QIAGEN Genomic-tip 100/G column with
4 mL
of Buffer QBT.
26 Pour the DNA in G2 buffer through the equilibrated column and allow it to ow through with just
gravity.
27
Once all the lysate has passed through, wash the column with
8 mL
of Buffer QC.
28
Repeat the wash with another
8 mL
of Buffer QC.
29
Place the column over a clean 50-mL Falcon tube, and elute the genomic DNA with
5 mL
of Buffer QF, pre-warmed to
55 °C
.
30
Allow the eluate to cool down to
Room temperature
.
31
Add
3.5 mL
of isopropanol to the eluted DNA and mix by inverting the tube 10 times.
32
Incubate the tube at
-20 °C
for at least 3 hours, or
Overnight
.
33
Centrifuge the tube at
3500 x g, 4°C, 00:45:00
.
34 Discard the supernatant without disturbing the pellet.
35
Add
4 mL
of ice-cold 70% ethanol to the pelleted DNA and invert the tube 10 times.
36
Centrifuge at
3500 x g, 4°C, 00:10:00
.
15m
45m
10m
Column cleanup
Final precipitation
1h 10m
protocols.io | https://dx.doi.org/10.17504/protocols.io.14egn2dnpg5d/v1 August 9, 2023 8/8
Note
Tip: If using a swinging bucket centrifuge the DNA will pellet at the base of the tube and be
easy to locate and resuspend. If using a xed angle, mark the side of the tube that faces
outwards in order to locate the pellet for washes and elution.
37 Discard the supernatant without disturbing the pellet. Use sterile wipes to dry the tube walls,
being careful not to disturb the pellet.
38
Resuspend the DNA in
100 µL
of TE buffer and incubate at
Room temperature
,
typically
Overnight
.
39 Transfer the DNA into a nuclease-free 1.5-mL tube (DNA LoBind tube preferred) using a wide-
bore tip, and store at
4 °C
.
Note
Tip: Often, waiting a further
48:00:00
before quantifying on Nanodrop and Qubit will
allow the DNA to further relax and yield the most accurate results
40 Carry samples forward to BluePippin size selection, if available. This gel separation will retain
DNA fragments greater than 15 kb and discard any residual contamination still evident on a
Nanodrop trace. For these benets, expect 50-70% loss of DNA.
Protocol references
Citations
1. Chekan, J. R. et al. Scalable Biosynthesis of the Seaweed Neurochemical, Kainic Acid. Angew Chem Int Ed Engl 58,
8454–8457 (2019).
2. Nanopore, Arabadopsis Leaf gDNA. https://community.nanoporetech.com/extraction_method_groups/plant-leaf-gDNA
15m
