wet-lab-documentation/EZ_nuclei_prep.docx.txt at master · corbett-lab/wet-lab-documentation · GitHub

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Nuclei extraction


Nuclei Isolation:
        Before starting the protocol begin cooling down a bucket centrifuge to 4C.
Wash flies
1. Collect 2-4 mLs of flies into 15mL Falcon tube.
2. Wash flies with 2-3x volume of 50% Bleach
3. Wash with 1-2x volume of sterile mqH2O
4. Wash with 1-2x volume of 80% EtOH
5. Wash with 1-2xvolume of sterile mqH2O
6. Wash with 1-2xvolume of ice cold 1xPBS
Lysis
1. Add 4mLs of ice cold Nuclei EZ Lysis buffer to falcon tube and transfer contents to dounce homogenizer
2. Place pestle into mortar and cover the opening with two strips of parafilm to prevent splashing.
3. Homogenize thoroughly taking care to not release the pestle when cavitating or YOU WILL BREAK THE GLASS.
4. Incubate on ice for 5 minutes.
5. Place 40 um cell strainer on 50mL falcon tube and strain homogenate through 2-3x.
6. Pour homogenate into clean 15mL falcon tube.
Isolation
        The following steps are performed exactly as the Nuclei EZ lysis protocol
1. Centrifuge homogenate at 500xg at 4C for 5 minutes.
2. Discard supernatant carefully to not disturb pellet.
3. Wash nuclei with 4mL of ice cold Nuclei EZ lysis buffer by first adding 500uL then vortexing. Then add remaining 3.5mLs. Set on ice for 5 minutes.
4. Centrifuge at 500xg at 4C for 5 minutes.
5. Discard supernatant and set pellet on ice.
6. Resuspend pellet in 200uL of ice cold Nuclei EZ storage buffer by gently pipetting.
7. Transfer final suspension to a tube for storage at -80C.
DAPI Staining QC
1. Gently mix nuclei suspension and transfer 20-50uL of suspension to 1.5mL tube.
2. Centrifuge tube at 300xg or slower for 5 minutes.
3. Discard supernatant.
4. Resuspend pellet in 1mL of DAPI stain and incubate for 15 minutes.
5. Centrifuge at 300xg for 5 minutes to pellet nuclei.
6. Discard supernatant.
7. Resuspend pellet in original volume of storage buffer.
8. Pipette 2-5uL of DAPI stained nuclei onto a slide and place a coverslip over sample.
9. Use nail polish to seal the coverslip to the slide.
10. Place finished slide into an opaque container in the fridge. Slides should be good for up to a week. DAPI stained nuclei can be stored in freezer indefinitely?
11. Use a widefield microscope to visualize nuclei.