Rat Liver Nuclei Prep
1. Obtain freshly excised livers, maintain in ice cold solution X with PMSF added just before use.
2. Clean livers of connective tissues and fat if from older rats.( Mince on an ice-cold plate with a razor blade. Transfer minced tissue to 2 volumes of solution X. ) We now just add 2 vols. of X and then homogenise directly.
3. Homogenise livers until homogeneous (use our new homogeniser at speed 4-5 for pulses of about 15sec, check for cell breakage under the microscope, until nuclei are seen.)
4. Filter through 4 layers of cheesecloth.
5. Add 2X vols. of solution Y, invert to mix.
6. Add approximately 30ml of homogenate to a Beckman ultra-centrifuge tube already containing 5mls of Y, pour on reasonably carefully. Balance tubes in pairs in buckets.
7. Spin at 22K for 2 hours at 4°C in the SW 28.
8. Remove plug of lipid from top of tube using a spatula. Invert tube, wipe walls with tissue. Resuspend the nuclear pellet in solution X using 2.2ml per 100ml of homogenate.
9. Count a 1/500 dilution of nuclei using Hoechst stain in a haemocytometer.
10.Quick freeze in liquid N2, store at -70°C.
Solutions
• Solution X 250mM sucrose
• 1mM DTT
• 1X Buffer A salts
• 0.5mM spermidine 2.5mls 100mM per 500mls
• 0.2mM spermine 1ml 100mM per 500mls
• 1µg/ml apoprotinin Add protease inhibitors after resuspending.
• 1µg/ml leupeptin 0.5mls A/L mix per 500mls
• 1mM PMSF 5mls 100mM in ETOH
• Solution Y same as solution X only with 2.3M sucrose
• Buffer salts A for solutions X and Y 5X per 500mls
• 80mM KCl 50mls 4M
• 5mM EDTA 125mls 100mM
• 15mM PIPES pH 7.4 11.34 g
• 15mM NaCl 2.19 g
