This article describes a method for isolating and purifying intact nuclei from a crude nuclei fraction using a multi-step discontinuous density gradient. The process involves centrifugation and careful layering of iodixanol solutions to achieve a clear separation of intact nuclei.
To isolate and purify intact nuclei, begin by taking a crude nuclei fraction in a tube. The fraction contains intact nuclei along with broken nuclei fragments and debris.
Mix this fraction with a suitable density gradient medium such that it forms a suspension of the lowest concentration.
Now, layer solutions of increasing densities of the same gradient medium beneath the pre-prepared crude nuclei fraction to generate a multi-step discontinuous density gradient. This step allows the crude nuclei fraction to form a band at the top of the gradient.
Next, centrifuge the tube at a low speed.
Based on the differences in shape and mass, intact nuclei migrate differently compared to debris and nuclear fragments. This results in intact nuclei forming a separate band.
After centrifugation, locate the distinct band consisting of intact nuclei at the interface of density gradient solutions.
Using a pipette, aspirate the top layers of the gradient to facilitate access to the pure nuclei band. Collect the nuclei-containing band into a fresh tube.
Visualize these nuclei under a light microscope. Intact nuclei appear perfectly round with an intact nuclear membrane surrounding them.
Finally, store the pure nuclear fraction for further analysis.
Add 200 microliters of 50% iodixanol solution to the nuclei for a final concentration of 25% iodixanol and mix 10 times with the pipette set to 300 microliters.
Then, add 300 microliters of 29% iodixanol solution under the 25% mixture using a P1000 fine tip to avoid mixing the layers. Add 300 microliters of 35% iodixanol solution under the 29% mixture with a P1000 fine tip. Then, slowly remove it.
Place the samples in a swinging bucket centrifuge and spin them for 20 minutes at 3,500 x g at 4 degrees Celsius with the brake off.
Gently remove the samples without shaking and observe them under light. A clear white band of 95% pure nuclei should be visible between the second and third layer.
To isolate the nuclei, aspirate the top layers to expose the white nuclei band at the interface. Collect the nuclei band in a 200-milliliter volume and transfer it to a fresh tube. Then, filter it with a 20-micrometer filter.
Check the nuclei under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. They should be round, and the nuclear membrane should not be distorted.
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