MNase Digestion

• injecting the rats with sodium pentobarbitol
• transcardial perfusion 100ml/2min Saline nitride heparine for each rat
• transcardial perfusion with 400ml/4min 1% phospahte buffered paraformaldehyde
• Dissect rat adrenal gland and put in PBS on ice and tranfer to the lab
• Separate medulla in PBS (the tissues were in PBS around 60 min)
• 4 medullas in Dounce homogenizer in 1ml PBS,
• Quenching by adding with 1/20 of stock Glycine (2.5M), final concentration is 125mM
• homogenize the tissues and let the tube sit for 5 min at RT
• Spinning down the cells at 16000rcf, supernatant is removed
• The cell pellet is resuspended in 1ml Nucleus Isolation Buffer (NIB) (10mM HEPES at pH 7.8, 2mM MgoAc2, 0.3M sucrose, 2mM CaCl2, 1%Triton-X) let the tube sit for 10 min at RT
• Spinning down the cells at 1000rcf, supernatant is removed
• resuspend the cells in 1ml NIB
• Spinning down the cells at 1000rcf, supernatant is removed
• resuspending the cells in 500 µl in NIB
• prewarm the cells ar 37°C form 1 min
• Prepare 2 MNase digestion reaction (total volume 250µl each), MNase stock concentration : 20U/µl diluted to 2 U/µl
• 1: add 5µl of 2U/µl to get 10U, 10min, RT
• 2: add 2.5µl of 20U/µl to get 50U, 10min, RT
• Stop the reaction with 10 µl EDTA(500mM), the final concentration was 20mM
• Add 6.25µl of 20%SDS (final concentration 0.5%)
• Adding 2.5µl of Proteinase K 20mg/µl) (final concentration 0.2 µg/l)
• Incubate at first 55 C for 1-2 hours and then overnight at 65 C
• add 2.4µl RNAse (final concentration 50µg/ml) for 20min at 37° C
• DNA Purification with just Phenol:cholorophorm:isoamyl and Ethanol precipitation