TITLE: Annonaceae DNA extraction protocol from silicagel dried and herbarium preserved leaves
AUTHORS: Vincent Soulé, Thomas LP Couvreur, Cedric Mariac

[DESCRIPTION]
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.

[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.

[STEPS]
SECTION: Leaf grinding
1. 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).


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

SECTION: Lysis buffer preparation and lysis
2. 
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 (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)
SECTION: Lysis buffer preparation and lysis
2.1. 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   at 1 mg/mL then vortex.                                                                                                  
Add 50 µL of    then vortex.
Add LBmix to adjust volume to 50 mL and put solution at 65 °C
Final volume 50 mL 
2.2. 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 180 min for silicagel preserved samples; for herbarium sampled leaves place at 65 °C for a recommended 360 min to 480 min. 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  1440 min  at  -20 °C 
SECTION: Chloroform DNA Isolation
3. 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 2 min  to 10 min
SECTION: Chloroform DNA Isolation
3.1. Centrifuge 4000 rpm, 10 min

Prepare in a new rack of 48 2mL tubes. 
3.6. 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.
SECTION: Chloroform DNA Isolation
3.3. Add 10 µL of    at 0.5 mg/mL
Place the rack with the samples at 37 °C  for 30 min .

SECTION: Chloroform DNA Isolation
3.4. 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.


SECTION: DNA precipitation
4. Add 360 µL of    at  -20 °C  
Add 60 µLof   pH=5 at 4 °C 
Slowly shake the tubes and then place the rack at -20 °C  for 120 min  to  
SECTION: Washing DNA
4.2. Add 700 µL of    in each tube to wash pellet.
SECTION: DNA precipitation
4.1. Centrifuge 14000 rpm, 10 min, -20 °C
 
Slowly remove liquid phase using a pipette with P1000, be aware to not pipette DNA pellet.
SECTION: Washing DNA
4.3. Centrifuge 14000 rpm, 10 min, -20 °C
Remove liquid phase with P1000, be aware to not pipette DNA pellet.
Let dry for 15 min at Room temperature.

SECTION: DNA elution
5. Depending on DNA pellet size, add 60-100 µL of  , shake tubes. 
  
Eluate    at 4 °C 
SECTION: DNA quantification
6. 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.

 

SECTION: Expected result
9. 
 


SECTION: DNA elution
5.1. Tubes can be stored for 2880 min at 4 °C before quality check and quantification.
SECTION: DNA quality check on gel
7. To the 4 µL left add 6 µL  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 40 min  on TAE 0.5X.

Put gel 15 min  in 1X   
Place gel in imaging machine. Turn on UV light, take a picture.
SECTION: DNA conservation
8. 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.

SECTION: Chloroform DNA Isolation
3.2. 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.
SECTION: Chloroform DNA Isolation
3.5. Centrifuge 4000 rpm, 10 min
During centrifugation, prepare and name new 2ml eppendorf safe lock tubes.
SECTION: Leaf grinding
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.