Problems with nucleus isolation and 293 cells - Nucleus seems to disrupt. What to do? (Oct/21/2008 )

Hi

I am trying to isolate nucleus from 293 cells for flow cytometry analysis and PCR (analysis of nucleus DNA). The problem is that the nucleus disrupts and all i end up with is a clump of DNA after step 5 in the protocol below (i can see the nuclei in microscope after the first lysis step).

Protocol i use:

Procedure for Suspension Cell Lines:
1. Grow cells in tissue culture flasks (15 ml per
75 cm2 flask) to desired cell density.

2. Harvest cells as follows. Transfer each culture
into a separate 15 ml centrifuge tube and
centrifuge at 500 x g for five minutes at 4 °C.
Carefully aspirate the supernatant and set the cell
pellet on ice.

3. Wash cells in 10 ml of ice cold Dulbecco’s
Phosphate Buffered Saline (PBS) as follows.
Vortex cell pellet briefly. Add 1 ml cold PBS and
vortex briefly at moderate to high speed to
completely suspend cells. Add remaining 9 ml of
PBS, mix and set on ice. Collect cells by
centrifugation as in step 2. Carefully aspirate clear
supernatants and set cell pellets on ice.

4. Lyse cells in 4 ml of ice cold Nuclei EZ lysis buffer
as follows. Vortex pellet briefly. Add 0.5 ml cold
Nuclei EZ lysis buffer and vortex briefly at
moderate to high speed to completely suspend
cells. Add the remaining 3.5 ml of Nuclei EZ lysis
buffer, mix well and set on ice for 5 minutes.

5. Collect the nuclei by centrifugation at 500 x g for
five minutes at 4 °C. Carefully aspirate the clear
supernatant from each tube and set the nuclei
pellet on ice. Note: The supernatant contains
cytoplasmic components and can be saved for
later analysis or use.

6. Resuspend and wash nuclei in 4 ml of ice cold
Nuclei EZ lysis buffer as follows. Vortex nuclei
pellet briefly. Add 0.5 ml cold Nuclei EZ lysis
buffer and vortex briefly at moderate to high speed
to completely suspend nuclei pellet. Add the
remaining 3.5 ml of Nuclei EZ lysis buffer, mix well
and set on ice for 5 minutes.

7. Collect washed nuclei by centrifugation as in
step 5. Carefully aspirate the clear supernatant
and set the nuclei pellet on ice.

8. Resuspend each nuclei pellet in ice cold PBS

Nuclei EZ lysis buffer: TricHCl 10 mmol/l, KCl 60 mmol/l, EDTA 1 mmol/l + protease inh. and 0,5 % Tergitol P-40)

Any suggestion what could be wrong? All the equipment i use is ice cold. One of my lab-partners use HeLa cells and the procedure works well for him.

the Nuclei ez that you are using has Tris, KCl and Tergitol p-40....but doesn't have Na.Deoxycholate to break and lyse the nuclei....so I'm not sure why your nuclei are lysed....the membrane must break to release cytoplasm by your buffer, but not nuclei !!

next time really make sure that you carry out all steps on ice....the clump you get is genomic DNA. it happens to me sometimes when I use RIPA buffer...if I don't carry out the procedure on ice it'll give me clumps.

there are many protocols for nuclei isolation, I know a good book called Cell Biology protocols by Robin Harris from Willey. it has hundreds of protocols.

also Dubleco's PBS is called DPBS...must be slightly different.


what we do is by Digitonin. but we isolate Mitochondria...you can use this method up to the step for Nuclei isolation:

Mitochondrial DNA book by William C Copeland, Methods in Molecular Biology, Volume 197, pages 351-355

Digitonin solution:
- 325mM Digitonin
- 2.5 mM EDTA
- 250mM Mannitol
- 17mM MOPS, pH7.4
2.5X Mannitol-Sucrose buffer:
- 525mM Mannitol
- 175mM Sucrose
- 12.5mM EDTA
- 12.5mM Tris-HCl, pH7.5
Organelle buffer:
- 20mM HEPES, pH7.6
- 1mM EDTA
- 5mM DTT
- 300mM KCl
- 5% Glycerol
PROCEDURE:
- Grow at least 10 million cells for each cell type. The number of cells required will depend on the number of mitochondria per cell. For HeLa cells, this is approximately three 75-cm2 flasks at near confluence
- Dislodge cells from monolayers with Trypsin and collect each cell type into a single pellet by centrifugation at 1000g for 10min
- resuspend each cell pellet in ice-cold digitonin solution for 80s.pipette cells on ice until no clumps are observed
- Add the lysed cell mixture to 2.5X Mannitol-sucrose buffer for a final strength of 1X (210mM Mannitol, 70mM Sucrose, 5mM EDTA, 5mM Tris-HCl, pH 7.5)
- Centrifuge the ice-cold suspension at 4 degrees Celcius for 10min at 800g to pellet nuclei
- save the supernatant, resuspend the pelleted nuclear material in 1X Mannitol-Sucrose, and repeat the centrifugation. Repeat this step 3 additional times.
- Combine the saved supernantant and centrifuge at 800g for 10min to pellet any remaining nuclei and very carefully draw off the resulting supernatant. Save the nuclei on ice
- centrifuge the new supernatant at 10,000g for 20min to pellet mitochondria. You should see a small pellet
-Decant supernatant carefully and save (this is cytosolic fraction) add 5ul/ml protease inhibitors
- resuspend isolated mitochondria and nuclei in organelle buffer + 5ul/mlprotease inhibitors (add just before use)
- Pulse-sonicate (1s) the resuspended organelles on ice
- centrifuge the sonicated organelles at 5000g for 10min to pellet remainin cell debris, and carefully draw off the supernatant
- determine protein concentrations for each sample by Bradford assay