2
edits
Changes
updated
* Dissect 4 rat adrenal gland and put in PBS
* injecting the rats with sodium pentobarbitol
* Separate medulla in PBS
* transcardial perfusion 100ml/2min Saline nitride heparine for each rat
* Homogenized 4 medulla in NIB
* 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)
* Quenching with 1/20 of stock Glycine (2.5M), final concentration is 125mM, 10min, RT
* 4 medullas in Dounce homogenizer in 1ml PBS,
* Spinning down the cells at low speed, supernatant is removed
* Quenching by adding with 1/20 of stock Glycine (2.5M), final concentration is 125mM
* The cell pellet is resuspended in 500microliter Nucleus Isolation Buffer (NIB) (10mM HEPES at pH 7.8, 2mM MgoAc2, 0.3M sucrose, 2mM CaCl2, 1%Triton-X)
* homogenize the tissues and let the tube sit for 5 min at RT
* Washing the cells two times with NIB
* Spinning down the cells at 16000rcf, supernatant is removed
* Prepare 2 digestion reaction (total volume 500 microliter):
* 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
* 1: 250 microliter of cells, 250 microliter NIB, 2U Mnase (20U/Microliter), 10min, RT
* Spinning down the cells at 1000rcf, supernatant is removed
* 2: 250 microliter of cells, 250 microliter NIB, 20U Mnase (20U/Microliter), 10min, RT
* resuspend the cells in 1ml NIB
* Stop the reaction with EDTA(500mM), the final concentration was 20mM
* Spinning down the cells at 1000rcf, supernatant is removed
* Addin 25microliter of 0.5%SDS
* resuspending the cells in 500 µl in NIB
* prewarm the cells ar 37°C form 1 min
* Adding 5microliter of Proteinase K
* Prepare 2 MNase digestion reaction (total volume 250µl each), MNase stock concentration : 20U/µl diluted to 2 U/µl
* Incubate at first 55 C and then overnight at 65 C
* 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
* DNA Purification with just Phenol:cholorophorm:isoamyl and Ethanol precipitation