Dec 05, 2023
Annonaceae DNA extraction protocol from silicagel dried and herbarium preserved leaves
- Vincent Soulé1,
- Couvreur LP Thomas1,
- Cedric Mariac1
- 1DIADE, Univ Montpellier, CIRAD, IRD, Montpellier, France
Protocol Citation: Vincent Soulé, Couvreur LP Thomas, Cedric Mariac 2023. Annonaceae DNA extraction protocol from silicagel dried and herbarium preserved leaves. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorqx9v4o/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: January 18, 2023
Last Modified: December 05, 2023
Protocol Integer ID: 75464
Keywords: DNA extraction, NGS extraction, MATAB, CTAB, aDNA, Illumina sequencing, Angiosperm, magnoliales, herbarium
Funders Acknowledgement:
ERC Consolidator
Grant ID: 865787
Disclaimer
This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 865787)
Abstract
This protocol is used for DNA extraction of samples from the tropical plant family Annonaceae for leaves dried using silicagel or sampled from herbarium sheets. This protocol is made for generation of small to long fragments (depending quality of sample) to prepare NGS libraries.
This protocol is designed to extract DNA in batches of 48 samples, but this can also be undertaken in 2 times 48 (96) samples.
Image Attribution
Art work by Vincent Soulé
Guidelines
Work in a clean environment to avoid contamination, use as much as possible filter tips, wear gloves and lab coat. Wash work space and pipettes before and after use, with DNAaway and DNAse away.
Manipulate with extreme caution rare or old samples.
Materials
2mL Screw tube
2mL secure lock ependorf tube
REF MATAB
Protocol materials
1M TE buffer (1M Tris-HCl, 0.1M EDTA, pH 8.0)
Step 5
AgaroseCatalog #A5304
Step 7
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
Step 2.1
RNase A Solution, 4mg/mlPromegaCatalog #A7973
Step 3.3
70% alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #793213
Step 4.2
Gel Red Nucleic Acid Gel StainBiotiumCatalog ##41003
Step 7
Na acetate 3M pH 4.8 or 5.2
Step 4
Isopropanol
Step 4
Proteinase KLife TechnologiesCatalog #17916
In 2 steps
Safety warnings
Always work under an extractor hood when manipulating of Chloroform, DTT or Isopropanol
Leaf grinding
Leaf grinding
Prepare 48 2 mL Screw-Top tubes in rows of 8 in a 96 well rack. Add one 1/4'' ceramic beads (MP Biomedical REF 116540422).
Add leaf sample inside the tube using clean tweezers. The leaf samples can be between 1x1 cm and 3x3 cm in size. Closes the tubes.
Grind samples using a MP FastPrep grinder, twice for 00:00:40 at 4m/second speed with a 2-minute pause in between each grind as not to over heat the samples.
Equipment
FastPrep-24™ 5G
NAME
grinder
TYPE
MP Biomedicals™
BRAND
116005500
SKU
LINK
24 samples
SPECIFICATIONS
4m
Lysis buffer preparation and lysis
Lysis buffer preparation and lysis
Lysis buffer LB needs to be freshly made the day of the extraction using a previously made LBmix + MATAB, Proteinase KLife TechnologiesCatalog #17916 and DTT (DL-Dithiothreitol) (final concentration 1mM).
Preparation of LBmix (1000 mL ) for 1000 samples:
1000 mL miliQ water Final concentration
9.31 g EDTA 25 millimolar (mM)
15.76 g Tris-HCL 100 millimolar (mM)
81.82 g NACl 1.4 Molarity (M)
Preparation of LB for 48 samples:
In a 50mL tube add 2 g of MATAB + 20 ml of LBmix Final concentration 4 Mass / % volume
Disolve MATAB in 65 °C water bath and vortex (aprox 10 min).
Add 250 µL of Proteinase KLife TechnologiesCatalog #17916 at 1 mg/mL then vortex.
Add 50 µL of DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632 then vortex.
Add LBmix to adjust volume to 50 mL and put solution at 65 °C
Final volume 50 mL
Using a p1000, add to each grinded sample 1 mL of still hot LB; close the tube then vortex each sample until the leaf powder is totally disolved.
Place the rack at 65 °C for a minimum of 03:00:00 for silicagel preserved samples; for herbarium sampled leaves place at 65 °C for a recommended 06:00:00 to 08:00:00 . Lysis can be done over night.
Shake the rack/tubes every 30 minutes during lysis.
17h
If needed, the process can be stopped after lysis, and the tubes can be conserved up to 24:00:00 at -20 °C
1d
Chloroform DNA Isolation
Chloroform DNA Isolation
For herbarium samples, limit to one round of chloroform isolation.
After the lysis step, let the samples get to room temperature.
Under an extractor hood, add 700 µL of 24:1 Chloroform : Isoamyl alcohol. Close the tube and shake vigorously by inversion the rack for 00:02:00 to 00:10:00
12m
Centrifuge 4000 rpm, Room temperature, 00:10:00
Prepare in a new rack of 48 2mL tubes.
10m
Under an extractor hood, transfer 8 by 8, using a multichannel pipette, 800 µL of the aqueous supernatant phase into the new 2mL tubes. Avoid pipetting the interphase pellet.
10m
Add 10 µL of RNase A Solution, 4mg/mlPromegaCatalog #A7973 at 0.5 mg/mL
Place the rack with the samples at 37 °C for 00:30:00 .
30m
Under an extractor hood, proceed with the second chloroform cleaning step. Add 700 µL of 24:1 Chloroform Isoamyl alcohol; close tubes and shake the rack of tubes.
10m
Centrifuge 4000 rpm, Room temperature, 00:10:00
During centrifugation, prepare and name new 2ml eppendorf safe lock tubes.
10m
Under an extractor hood, transfer 8 by 8 using a multichannel pipette, 600-800 µL of the aqueous supernatant phase into the new 2mL tubes. Avoid pipetting the interphase pellet.
5m
DNA precipitation
DNA precipitation
2h
Add 360 µL of Isopropanol at -20 °C
Add 60 µL of Na acetate 3M pH 4.8 or 5.2 pH=5 at 4 °C
Slowly shake the tubes and then place the rack at -20 °C for 02:00:00 to Overnight
2h
Centrifuge 14000 rpm, -20°C, 00:10:00
Slowly remove liquid phase using a pipette with P1000, be aware to not pipette DNA pellet.
10m
Washing DNA
Washing DNA
25m
Add 700 µL of 70% alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #793213 in each tube to wash pellet.
10m
Centrifuge 14000 rpm, -20°C, 00:10:00
Remove liquid phase with P1000, be aware to not pipette DNA pellet.
Let dry for 00:15:00 at Room temperature .
25m
DNA elution
DNA elution
Depending on DNA pellet size, add 60-100 µL of 1M TE buffer (1M Tris-HCl, 0.1M EDTA, pH 8.0) , shake tubes.
10 rpm, Room temperature
Eluate Overnight at 4 °C
20m
Tubes can be stored for 48:00:00 at 4 °C before quality check and quantification.
2d
DNA quantification
DNA quantification
55m
Transfer 6 µL of each samples to a 96 well PCR plate.
Use 2 µL for DNA quantification using TECAN spark or nanoquant with nanoquant 16 holes plate; or quantify one by one using Nanodrop.
Equipment
SPARK
NAME
Microwell plate reader
TYPE
TECAN
BRAND
SPARK
SKU
LINK
DNA quality check on gel
DNA quality check on gel
55m
To the 4 µL left add 6 µL 1.5X red blue yellow loading buffer.
Prepare 1% of AgaroseCatalog #A5304 gel with TAE 1X.
Add your samples and:
+ promega 100pb dna ladder
+ promega 2.5kb lambda eco R1 hind3 dna ladder on gel well
Then proceed with gel electrophoresis at 135V 00:40:00 on TAE 0.5X.
Put gel 00:15:00 in 1X Gel Red Nucleic Acid Gel StainBiotiumCatalog ##41003
Place gel in imaging machine. Turn on UV light, take a picture.
55m
DNA conservation
DNA conservation
55m
Extracted DNA needs to be stored at -20 °C .
For long term conservation, transfert to barcoded screw-top tube on 96 rack.
For the GLOBAL projet we used Thermo Scientific™ Matrix™ 0.5mL 2d barcoded.
Expected result
Expected result
Expected result
Within the GLOBAL project, around 3600 DNA extractions were undertaken, some on silicagel leaves others on herbarium preserved leaves.
For silicagel dried samples we extracted 1050 specimens, with a max concentration of 979 ng/ul and a minimum of 0.9 ng/ul for a total elution volume of 100 ul. On average we had 213 ng/ul (Standard error: 205.8).
For herbarium preserved leaves 2600 samples were extracted: the highest concentration was 1088ng/ul and the lowest was 0,1 ng/ul for a total elution volume of 60 ul. On average we had 230 ng/ul per extraction (Standard error: 221.8).
Concentrations below 10 ng/ul were rarely used to sequenced or frequently failed.
DNA size ranged between 100pb-2.5kb, but longer fragments were also possible.
Protocol references
Part of this protol follows:
Couvreur TLP, Helmstetter AJ, Koenen EJM, Bethune K, Brandão RD, Little SA, Sauquet H, Erkens RHJ (2019) Phylogenomics of the Major Tropical Plant Family Annonaceae Using Targeted Enrichment of Nuclear Genes. Frontiers in Plant Science 9. https://doi.org/10.3389/fpls.2018.01941