Our optimised protocol to produce a large quantity of high quality long DNA molecules, suitable for PacBio sequencing

 

Materials:  

Centrifuge

Douncer

Qubit

 

Reagents and Solutions: 

-All buffers must be kept on ICE

-All buffers must be MADE FRESH.

-For all buffer containing PI, prepare only the volume needed.

-For 1 sample, you need:

19ml+ 5ml Extraction Buffer 1

1ml Extraction buffer 2

1,3ml extraction buffer 3

 

Extraction Buffer 1        for 19ml:

10mM HEPES pH=7.6             200µL 1M                  

0.5M sucrose                          4.75m l 2M

5mM KCl                40µl 500mM

10mM MgCl            200µl 1M

5mM EDTApH=8                          2mL 50mM

1x Proteinase Inhibitor (PI)     200µl 100x

H20 to volume                          11.61ml

 

Extraction Buffer 2                 for 5mL:        

0.25M sucrose                                630µl  2M        

10mM Tris-HCl pH 8                      50µl  1M        

10mM MgCl2                                  50µl  1M    

1% Triton X-100                             250µl      20%    

5mM BME                                      1.75µl  14.3M    

1x Proteinase Inhibitor (PI)      50µl  100x    

H20 to volume                                  3.968ml    

 

Extraction Buffer 3                    for 10 ml:    

1.7M sucrose            8.5 ml 2M

10mM Tris-HCl pH 8        100 μl 1M

0.15% Triton X-100        75 μl 20%

2mM MgCl2            20 μl 1M

5mM BME                3.5 μl 14.3M

1x Proteinase Inhibitor (PI)    100µL 100x

H20 to volume            1.2ml

 

Nuclei Lysis Buffer

(NOT IN ICE)            For 1ml:

50mM Tris-HCl pH 8        50µl  1M

10mM EDTA            200µl 50mM

1% SDS                200µl 5%

1x Proteinase Inhibitor (PI)    10µl 100x

H20 to volume            540µl

 

Risks and security :

Work under the hood 

 

Protocol : 

Nuclei isolation 

Use 1.5 ml normal eppendorf tube for all steps

 

-Grind 0.5g of material in liquid N2 ( grind till' you have tea leaf size fragments as in a tea bag...)

- In 50mL falcon tube, re-suspend leaves fragments in 19 mL cold Extraction Buffer 1

- Keep 1mn on ice.

- On ice, homogenize in a 40 ml douncer tissue grinder (Wheaton, USA), 7 strokes with the loose pestle and 7 strokes with the tight pestle. (This is required in order to ensure that all nuclei are properly released).

- Recover the lysate by filtering through 4 layers of Miracloth into a fresh 50ml falcon tube.

- Rinse the douncer/pestle with 5 ml Extraction Buffer 1  and rinse filter.

- Spin down nuclei at 4000 g (speed) for 20 min at 4°C (better if you can use “swinging” rotor)

- Gently remove supernatant and re-suspend the pellet in 1ml of Extraction Buffer 2 (use a small and clean paint brush)

- Transfer the solution to 1.5 ml Eppendorf tube.

- Centrifuge at 12,000 g for 10 minutes at 4 degrees.

- Remove supernatant and re-suspend pellet in 300μl of Extraction Buffer 3.

- In a clean Eppendorf, add 1ml of Extraction Buffer 3.

-Take the 300 μl solution (resuspended pellet) from previous step and carefully layer it on top of the clean 1ml of Extraction Buffer 3.(note: If the solution looks too viscous , you can split in half)

- Spin for 1 hour at 16,000 g at 4 degrees. Make Nuclei Lysis Buffer and ChIP dilution buffer at this stage.

- Remove the supernatant (Note: if the pellet is really green, you can repeat the gradient step)

- Re-suspend the chromatin pellet in 500ml of Nuclei Lysis Buffer

- Add 20 ul RNase and leave 15 min at room T°

 

DNA recovery  

IMPORTANT: NEVER vortex (it will break the DNA).

Cut the bottom end of your tips to reduce friction and damage to the DNA.

Add 500µl phenol/chloroform/IAA extraction (25/24/1) pH=8, mix vigorously, centrifuge 10mns at full speed, recover supernatant,

add 500µl chloroform/IAA extraction, mix vigorously, centrifuge 1mn, recover supernatant,

 

add EtOH precipitation with 1/10 salt:

50μl NaAc  3M pH=5.2

500μ ethanol 95%

 

Precipitate at -20°C overnight

Spin at full speed 20 min.

Wash pellets with 500μl of 70% ethanol (leave DNA in EtOH if you need to send it for sequencing)

ⓒ by the Bucher laboratory