https://wiki.houptlab.org/api.php?action=feedcontributions&feedformat=atom&user=NabiMagnetoWiki - User contributions [en]2026-04-21T19:30:27ZUser contributionsMediaWiki 1.35.8https://wiki.houptlab.org/index.php?title=MNase_digestion&diff=3289MNase digestion2025-05-20T19:18:28Z<p>Nabi: Nabi moved page MNase digestion to MNase Digestion</p>
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<div>#REDIRECT [[MNase Digestion]]</div>Nabihttps://wiki.houptlab.org/index.php?title=MNase_Digestion&diff=3288MNase Digestion2025-05-20T19:18:28Z<p>Nabi: Nabi moved page MNase digestion to MNase Digestion</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 />
* 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>Nabihttps://wiki.houptlab.org/index.php?title=MNase_Digestion&diff=3287MNase Digestion2025-05-20T19:14:48Z<p>Nabi: Creating a page</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 />
* 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>Nabihttps://wiki.houptlab.org/index.php?title=Adrenal_Gland&diff=3286Adrenal Gland2025-05-07T21:10:19Z<p>Nabi: /* Rat Adrenal Gland Facts */ added note about cortex</p>
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<div><br />
Some papers on [https://houptlab.org/courses/adrenal adrenal gland and regulation of the medulla].<br />
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=Rat Adrenal Gland Facts=<br />
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''From Tomlinson 1987 PMID 3601067''<br />
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Adrenal Gland: 13.2 mm<sup>3</sup><br />
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Adrenal Cortex: '''(NOTE: need to add number of cells)'''<br />
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Adrenal Medulla: 1.3 mm<sup>3</sup><br />
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Medulla by volume: 63% chromaffin (catecholamine) cells, 20% vasculature, 5% neural, 12% interstitial<br />
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Number of cells (unilateral):<br />
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* 5 x10<sup>5</sup> epinephrine cells (TH & PNMT)<br />
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* 2 x 10<sup>5</sup> norepinephrine cells (TH)</div>Nabi