3
edits
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* Dissect 4 rat adrenal gland and put in PBS
* Separate medulla in PBS
* Homogenized 4 medulla in NIB
* Crosslinking cells with 1% formaldehyde, 15min, RT
* Quenching with 1/20 of stock Glycine (2.5M), final concentration is 125mM, 10min, RT
* Spinning down the cells at low speed, supernatant is removed
* 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)
* Washing the cells two times with NIB
* Prepare 2 digestion reaction (total volume 500 microliter):
* 1: 250 microliter of cells, 250 microliter NIB, 2U Mnase (20U/Microliter), 10min, RT
* 2: 250 microliter of cells, 250 microliter NIB, 20U Mnase (20U/Microliter), 10min, RT
* Stop the reaction with EDTA(500mM), the final concentration was 20mM
* Addin 25microliter of 0.5%SDS
* RNase 5 microliter
* Adding 5microliter of Proteinase K
* Incubate at first 55 C and then overnight at 65 C
* DNA Purification with just Phenol:cholorophorm:isoamyl and Ethanol precipitation
* Separate medulla in PBS
* Homogenized 4 medulla in NIB
* Crosslinking cells with 1% formaldehyde, 15min, RT
* Quenching with 1/20 of stock Glycine (2.5M), final concentration is 125mM, 10min, RT
* Spinning down the cells at low speed, supernatant is removed
* 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)
* Washing the cells two times with NIB
* Prepare 2 digestion reaction (total volume 500 microliter):
* 1: 250 microliter of cells, 250 microliter NIB, 2U Mnase (20U/Microliter), 10min, RT
* 2: 250 microliter of cells, 250 microliter NIB, 20U Mnase (20U/Microliter), 10min, RT
* Stop the reaction with EDTA(500mM), the final concentration was 20mM
* Addin 25microliter of 0.5%SDS
* RNase 5 microliter
* Adding 5microliter of Proteinase K
* Incubate at first 55 C and then overnight at 65 C
* DNA Purification with just Phenol:cholorophorm:isoamyl and Ethanol precipitation