Annonaceae DNA extraction protocol from silicagel dried and herbarium preserved leaves

Vincent Soulé, Couvreur LP Thomas, Cedric Mariac

Published: 2023-12-06 DOI: 10.17504/protocols.io.5qpvorqx9v4o/v1

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.

Steps

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).

1.1.

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.

1.2.

Grind samples using a MP FastPrep grinder, twice for 0h 0m 40s at 4m/second speed with a 2-minute pause in between each grind as not to over heat the samples.

Equipment

Value	Label
FastPrep-24™ 5G	NAME
grinder	TYPE
MP Biomedicals™	BRAND
116005500	SKU
24 samples	SPECIFICATIONS

Lysis buffer preparation and lysis

Lysis buffer LB needs to be freshly made the day of the extraction using a previously made LBmix + MATAB, and DTT (DL-Dithiothreitol) (final concentration 1mM).

Preparation of LBmix (1000mL) for 1000 samples:

1000mL miliQ water Final concentration

9.31g EDTA 25millimolar (mM)

15.76g Tris-HCL 100millimolar (mM)

81.82g NACl 1.4Molarity (M)

2.1.

Preparation of LB for 48 samples:

In a 50mL tube add 2g of MATAB + 20 ml of LBmix Final concentration 4Mass / % volume

Disolve MATAB in 65°C water bath and vortex (aprox 10 min).

Add 250µL of at 1mg/mL then vortex.

Add 50µL of then vortex.

Add LBmix to adjust volume to 50mL and put solution at 65°C

Final volume 50mL

2.2.

Using a p1000, add to each grinded sample 1mL 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 3h 0m 0s for silicagel preserved samples; for herbarium sampled leaves place at 65°C for a recommended 6h 0m 0s to 8h 0m 0s. Lysis can be done over night.

Shake the rack/tubes every 30 minutes during lysis.

2.3.

If needed, the process can be stopped after lysis, and the tubes can be conserved up to 24h 0m 0s at -20°C

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 700µL of 24:1 Chloroform : Isoamyl alcohol. Close the tube and shake vigorously by inversion the rack for 0h 2m 0s to 0h 10m 0s

3.1.

Centrifuge 4000rpm

Prepare in a new rack of 48 2mL tubes.

3.2.

Under an extractor hood, transfer 8 by 8, using a multichannel pipette, 800µL of the aqueous supernatant phase into the new 2mL tubes. Avoid pipetting the interphase pellet.

3.3.

Add 10µL of at 0.5mg/mL

Place the rack with the samples at 37°C for 0h 30m 0s .

3.4.

Under an extractor hood, proceed with the second chloroform cleaning step. Add 700µL of 24:1 Chloroform Isoamyl alcohol; close tubes and shake the rack of tubes.

3.5.

Centrifuge 4000rpm

During centrifugation, prepare and name new 2ml eppendorf safe lock tubes.

3.6.

Under an extractor hood, transfer 8 by 8 using a multichannel pipette, 600-800µL of the aqueous supernatant phase into the new 2mL tubes. Avoid pipetting the interphase pellet.

DNA precipitation

Add 360µL of at -20°C

Add 60µLof pH=5 at 4°C

Slowly shake the tubes and then place the rack at -20°C for 2h 0m 0s to

4.1.

Centrifuge 14000rpm,-20°C

Slowly remove liquid phase using a pipette with P1000, be aware to not pipette DNA pellet.

Washing DNA

4.2.

Add 700µL of in each tube to wash pellet.

4.3.

Centrifuge 14000rpm,-20°C

Remove liquid phase with P1000, be aware to not pipette DNA pellet.

Let dry for 0h 15m 0s at Room temperature.

DNA elution

Depending on DNA pellet size, add 60-100µL of , shake tubes.

10rpm

Eluate at 4°C

5.1.

Tubes can be stored for 48h 0m 0s at 4°C before quality check and quantification.

DNA quantification

Transfer 6µL of each samples to a 96 well PCR plate.

Use 2µL for DNA quantification using TECAN spark or nanoquant with nanoquant 16 holes plate; or quantify one by one using Nanodrop.

Equipment

Value	Label
SPARK	NAME
Microwell plate reader	TYPE
TECAN	BRAND
SPARK	SKU

DNA quality check on gel

To the 4µL left add 6µL 1.5X red blue yellow loading buffer.

Prepare 1% of 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 0h 40m 0s on TAE 0.5X.

Put gel 0h 15m 0s in 1X

Place gel in imaging machine. Turn on UV light, take a picture.

DNA conservation

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

Citation

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.