https://wiki.houptlab.org/api.php?action=feedcontributions&feedformat=atom&user=SoheilaMagnetoWiki - User contributions [en]2026-06-10T05:18:24ZUser contributionsMediaWiki 1.35.8https://wiki.houptlab.org/index.php?title=MNase_Digestion&diff=3302MNase Digestion2025-07-22T21:43:15Z<p>Soheila: updated</p>
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<div>* injecting the rats with sodium pentobarbitol<br />
* transcardial perfusion 100ml/2min Saline nitride heparine for each rat<br />
* transcardial perfusion with 400ml/4min 1% phospahte buffered paraformaldehyde<br />
* Dissect rat adrenal gland and put in PBS on ice and tranfer to the lab <br />
* Separate medulla in PBS (the tissues were in PBS around 60 min)<br />
* 4 medullas in Dounce homogenizer in 1ml PBS, <br />
* Quenching by adding with 1/20 of stock Glycine (2.5M), final concentration is 125mM<br />
* homogenize the tissues and let the tube sit for 5 min at RT<br />
* Spinning down the cells at 16000rcf, supernatant is removed<br />
* 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<br />
* Spinning down the cells at 1000rcf, supernatant is removed<br />
* resuspend the cells in 1ml NIB <br />
* Spinning down the cells at 1000rcf, supernatant is removed<br />
* resuspending the cells in 500 µl in NIB<br />
* prewarm the cells ar 37°C form 1 min<br />
* Prepare 2 MNase digestion reaction (total volume 250µl each), MNase stock concentration : 20U/µl diluted to 2 U/µl<br />
* 1: add 5µl of 2U/µl to get 10U, 10min, RT<br />
* 2: add 2.5µl of 20U/µl to get 50U, 10min, RT<br />
* Stop the reaction with 10 µl EDTA(500mM), the final concentration was 20mM<br />
* Add 6.25µl of 20%SDS (final concentration 0.5%)<br />
* Adding 2.5µl of Proteinase K 20mg/µl) (final concentration 0.2 µg/l)<br />
* Incubate at first 55 C for 1-2 hours and then overnight at 65 C<br />
* add 2.4µl RNAse (final concentration 50µg/ml) for 20min at 37° C<br />
* DNA Purification with just Phenol:cholorophorm:isoamyl and Ethanol precipitation</div>Soheilahttps://wiki.houptlab.org/index.php?title=MNase_Digestion&diff=3290MNase Digestion2025-06-01T16:28:42Z<p>Soheila: test Soheila account</p>
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<div>* Dissect 4 rat adrenal gland and put in PBS<br />
* Separate medulla in PBS<br />
* Homogenized 4 medulla in NIB <br />
* (filter homogenate?)<br />
* Crosslinking cells with 1% formaldehyde, 15min, RT<br />
* Quenching with 1/20 of stock Glycine (2.5M), final concentration is 125mM, 10min, RT<br />
* Spinning down the cells at low speed, supernatant is removed<br />
* 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)<br />
* Washing the cells two times with NIB<br />
* Prepare 2 digestion reaction (total volume 500 microliter):<br />
* 1: 250 microliter of cells, 250 microliter NIB, 2U Mnase (20U/Microliter), 10min, RT<br />
* 2: 250 microliter of cells, 250 microliter NIB, 20U Mnase (20U/Microliter), 10min, RT<br />
* Stop the reaction with EDTA(500mM), the final concentration was 20mM<br />
* Addin 25microliter of 0.5%SDS<br />
* RNase 5 microliter<br />
* Adding 5microliter of Proteinase K<br />
* Incubate at first 55 C and then overnight at 65 C<br />
* DNA Purification with just Phenol:cholorophorm:isoamyl and Ethanol precipitation</div>Soheila