https://wiki.xenbase.org/xenwiki/index.php?title=Isolation_of_chromatin_from_egg_extract_and_histone_recovery_(Shechter_lab)&feed=atom&action=historyIsolation of chromatin from egg extract and histone recovery (Shechter lab) - Revision history2024-03-28T17:37:22ZRevision history for this page on the wikiMediaWiki 1.40.1https://wiki.xenbase.org/xenwiki/index.php?title=Isolation_of_chromatin_from_egg_extract_and_histone_recovery_(Shechter_lab)&diff=47218&oldid=previmported>Virgilio: moved Shechter Lab - isolation of chromatin from egg extract and histone recovery to isolation of chromatin from egg extract and histone recovery (Shechter lab)2011-11-15T19:42:49Z<p>moved <a href="/xenwiki/index.php?title=Shechter_Lab_-_isolation_of_chromatin_from_egg_extract_and_histone_recovery" class="mw-redirect" title="Shechter Lab - isolation of chromatin from egg extract and histone recovery">Shechter Lab - isolation of chromatin from egg extract and histone recovery</a> to <a href="/xenwiki/index.php?title=Isolation_of_chromatin_from_egg_extract_and_histone_recovery_(Shechter_lab)" title="Isolation of chromatin from egg extract and histone recovery (Shechter lab)">isolation of chromatin from egg extract and histone recovery (Shechter lab)</a></p>
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</td></tr></table>imported>Virgiliohttps://wiki.xenbase.org/xenwiki/index.php?title=Isolation_of_chromatin_from_egg_extract_and_histone_recovery_(Shechter_lab)&diff=47216&oldid=prev205.142.197.33: Created page with "Protocol submitted by VGP from David Shechter lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1810&tabId=0] Link to PDF: [http://www.shechterl..."2011-11-11T19:39:56Z<p>Created page with "Protocol submitted by VGP from David Shechter lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1810&tabId=0] Link to PDF: [http://www.shechterl..."</p>
<p><b>New page</b></p><div>Protocol submitted by VGP from David Shechter lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1810&tabId=0]<br />
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Link to PDF: [http://www.shechterlab.org/wp-content/uploads/2009/07/Chromatin-Isolation-from-Egg-Extract.pdf]<br />
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PROTOCOL: Sperm Chromatin Isolation / Histone Preparation<br />
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Recipes:<br />
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ELB-CIB (Egg lysis buffer – Chromatin Isolation Buffer) - 1X Egg Lysis Buffer, containing .25M Sucrose 1mM Spermidine 1mM Spermine 0.5mM EDTA<br />
5mM DTT 0.1% Triton-X 100 1X Protease Inhibitor Cocktail (Roche) 10mM Sodium Butyrate 1X Phosphatase Inhibitor Cocktails I & II (Roche)<br />
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ELB - 0.25M Sucrose 2.5mM MgCl2 50mM KCl 10mM Hepes-KOH pH 7.8 1mM DTT<br />
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Procedures/Steps:<br />
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1. Incubate sperm in egg extract (LSS, HSS, NPE, etc), use 10-100 μl/reaction, and 2000- 10,000 sperm/ μl. Flash freeze at time points or continue with unfrozen extract. For large-scale histone preparation: use 1-2ml of LSS extract and 10,000 sperm/ μl.<br />
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2. Add 900 μl of 1X ELB-CIB (ice-cold); mix thoroughly and incubate on ice for 10 minutes. Underlayer carefully with 200 μl of ELB-CIB containing 0.6M sucrose.<br />
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3. Spin in swinging bucket rotor at 4000g for 5-10 minutes at 4C. (In eppendorf, 6000RPM). There should be a visible white pellet at the bottom of the tube.<br />
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4. Carefully remove supernatant with pipet and respin briefly. Aspirate remaining supernatant with pipet.<br />
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5. Wash pellet 1X with 1ml ELB-CIB. (For histone prep, include 0.3M NaCl). Completely aspirate supernatant with pipet.<br />
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6. For gel analysis: Resuspend pellet in 1X Laemmli loading buffer. The DNA can get viscous when hot; cool before loading on SDS-PAGE gel.<br />
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7. For histone prep: Extract pellet in 0.4N H2SO4 for 2 hours or overnight. Precipitate extracted proteins in supernatant with 25% cold TCA for 30 minutes on ice, spin down and wash pellet with 100% cold acetone. Dissolve pellet in appropriate volume of water.</div>205.142.197.33