isolation and extraction of plant nuclei in plug

NIB Buffer : 200 ml : freshly prepared

A	B
Tris ph8	10 mM
EDTA	10 mM
KCl	80 mM
Sucrose	0.5 M
PVP 40	2 %
Spermine	1 mM
Spermidine	1 mM
H2O	qsp 200 ml

Adjust the Ph to 9.4 then filter at 0.22 µm

NIBT Buffer : 160 ml

A	B
NIB Buffer	160 ml
Triton X100	0.5%

NIBTM Buffer : 40 ml

A	B
NIBT Buffer	40 ml
2-Mercaptoethanol	0.75 %

Cell suspension Buffer : Can be stored for 1 year at 4°C.

A	B
Tris ph8	10 mM
EDTA	50 mM
NaCl	2 mM
H2O	Qsp 100ml

Lysis Buffer : Can be stored for 1 year at RT.

A	B
EDTA 0.5M	100 ml
N-Lauroylsarcosine	1 %

Putting a mortar in ice

Cool a mortar/pestle with liquid nitrogen until the bubbling stops.

Place a beaker in ice and add a magnetic stirrer.

Add 20 ml of NIBTM (10 ml/g of leaves) and stir gently

20mL

Grind 2g of frozen sample for without adding liquid nitrogen, until a fine powder is obtained (approx. 2 min)

2g 0h 2m 0s

Transfer the powder to the beaker and shake gently for 10 minutes in ice.

0h 10m 0s

Filter the mixture into a 50ml tube through autoclaved filters (2 cheese cloth + 2 Mira cloth) on a funnel (squeeze the filters at the end of filtration to recover more of the solution containing the nuclei).

Filter through a 40µm cell stariner into a new 50ml tube.

Pelleting the homogenate by centrifugation.(acceleration and deceleration at level 3)

800x g,4°C

Remove the supernatant and gently resuspend the pellet in ice (use a brush if the pellet does not recover).

Add 20ml of cold NIBTM

20mL

Pelleting the homogenate by centrifugation to remove residues and unlysed cells.

60x g,4°C

Filter the supernatant through a 40 µm cell sieve into a new 50 ml tube.

Pellet the nuclei by centrifugation.

800x g,4°C

Wash the pellet 3 times in NIBT buffer :

- Remove the supernatant

- Gently resuspend the pellet

- Add 40 ml of cold NIBT buffer `40mL` 



- Centrifuge `800x g,4°C` 

Make a final wash in 30 ml of cold NIB buffer.

800x g,4°C

Resuspend the last pellet in the residual buffer (approx. 200 µl) and transfer the homogenate to a 1.5 ml tube.

Centrifuge.

800x g,4°C

Remove the supernatant with a pipette and resuspend the pellet in an appropriate volume of cell suspension buffer :

60µl / plug

Adjust the number of plugs to be made according to the size of the pellet.

optional : microscopic observation

- Take an aliquot of 100 µl of suspension

- Stain with DAPI

- Observe the presence of nuclei

Use Low Melting agarose for a final agarose concentration of 0.8 %

Put a CHEF Disposable Plug Molds on ice

Melt agarose at 70°C for 5 min then equilibrate at 43°C for 5 min

0h 5m 0s 70°C

0h 5m 0s 43°C

Preheat the nuclei suspension to 43°C for 3min and then add the appropriate amount of 2% agarose (see table).

Mix gently with a wide-bore tip, avoiding bubbles.

0h 3m 0s 43°C

Immediately dispense 100µl of mixture per well using wide-bore tips.

Allow to polymerise for 15 minutes on ice.

0h 15m 0s On ice

Prepare a fresh proteinase K digestion solution by mixing 200 μl of proteinase K enzyme (20mg/ml) with 2.5 ml of lysis buffer in a 50 ml tube.

200µL 2.5mL

Transfer plugs to the 50ml tube containing Proteinase K digestion solution.

Incubate in thermomixer for 2 hours at 50 °C with intermittent mixing

Mixing cycle : 10 seconds at 450 rpm followed by 10 minutes at 0 rpm

2h 0m 0s 50°C

Screw a sieve caps onto the tube and empty the solution. Change the proteinase K Solution bath as before.

Incubate in thermomixer overnight at 50 °C with intermittent mixing

Mixing cycle : 10 seconds at 450 rpm followed by 10 minutes at 0 rpm

50°C

Prepare the wash solutions :

TE 10:50 (Wash Buffer)

TE 10:5 (For Rnase)

Empty the tube using a vent cap.

Rinse the plugs 3 times with 10ml of wash buffer.

Wash 2 times with 10ml wash buffer for 15 min at RT with gentle agitation (15 rpm) on a horizontal platform mixer.

15rpm

Rinse the plugs 3 times with 10ml of TE 10:5

Add 2.5ml of TE 10:5 and 50 µl of Rnase Solution

Incubate 1hour at 37°C with intermittent mixing

1h 0m 0s 37°C

Rinse the plugs 3 times with 10ml of Wash Buffer.

  ***NB : The plugs can be stored at 4°C in a wash buffer at this stage*** 

Wash 4 times with 10ml wash buffer for 15 min at RT with gentle agitation (15 rpm) on a horizontal platform mixer.

15rpm

Transfer the plug to a 1.5 ml tube with a sterile spatula

Melt the plug in a water bath at 70°C for 2 minutes

0h 2m 0s 70°C

Transfer the tube to a water bath at 43°C for 5 minutes

0h 5m 0s 43°C

Add 2µl of agarase and mix gently by rotating with the tip.

Incubate 45 minutes at 43 °C

0h 45m 0s 43°C

Place 10 ml of 1x TE Buffer in a 6 cm Petri dish.

Float a 0.1 μm dialysis membrane on the surface of the 1x TE Buffer. Place a cover on the Petri dish and let the membrane equilibrates for 15 minutes.

0h 15m 0s

Deposit the entire sample in the centre of the membrane using a wide-bore tip.

Place cover on the Petri dish and let the sample dialyze for 45 minutes at room temperature.

0h 45m 0s Room temperature

Transfer DNA to a 1.5 ml microfuge tube with a Wide Bore Tip.

Allow the DNA to resuspend overnight at RT then 2 days at 4°C before performing quality control.

Room temperature

Quantify your sample with a Qubit HS .

NB : Before quantification, sonicate the DNA aliquot for 10 min to obtain a more reliable result

Visualise 1 µL of sample to estimate the molecular weight. ( Tapestation or/and pipin pulse or/and Femto pulse )

QC results obtained on different plant species, using different technologies to estimate the size of the molecules.