https://wiki.xenbase.org/xenwiki/index.php?action=history&feed=atom&title=CSF_extract%2C_sara%27s_protocol_%28Stukenberg_lab%29 2026-09-03T13:21:10Z Revision history for this page on the wiki MediaWiki 1.44.2 https://wiki.xenbase.org/xenwiki/index.php?title=CSF_extract,_sara%27s_protocol_(Stukenberg_lab)&diff=47234&oldid=prev 2011-11-15T19:52:15Z

<p>moved <a href="/xenwiki/index.php/Stukenberg_Lab_-_Sara%27s_CSF_extract" class="mw-redirect" title="Stukenberg Lab - Sara&#039;s CSF extract">Stukenberg Lab - Sara&#039;s CSF extract</a> to <a href="/xenwiki/index.php/CSF_extract,_sara%27s_protocol_(Stukenberg_lab)" title="CSF extract, sara&#039;s protocol (Stukenberg lab)">CSF extract, sara&#039;s protocol (Stukenberg lab)</a></p> <table style="background-color: #fff; color: #202122;" data-mw="interface"> <tr class="diff-title" lang="en"> <td colspan="1" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td> <td colspan="1" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 19:52, 15 November 2011</td> </tr><tr><td colspan="2" class="diff-notice" lang="en"><div class="mw-diff-empty">(No difference)</div> </td></tr></table>

imported>Virgilio https://wiki.xenbase.org/xenwiki/index.php?title=CSF_extract,_sara%27s_protocol_(Stukenberg_lab)&diff=47233&oldid=prev 2011-11-15T15:42:20Z

<p>moved <a href="/xenwiki/index.php/CSF_Extract_(2)" class="mw-redirect" title="CSF Extract (2)">CSF Extract (2)</a> to <a href="/xenwiki/index.php/Stukenberg_Lab_-_Sara%27s_CSF_extract" class="mw-redirect" title="Stukenberg Lab - Sara&#039;s CSF extract">Stukenberg Lab - Sara&#039;s CSF extract</a>: added lab attribution</p> <table style="background-color: #fff; color: #202122;" data-mw="interface"> <tr class="diff-title" lang="en"> <td colspan="1" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td> <td colspan="1" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 15:42, 15 November 2011</td> </tr><tr><td colspan="2" class="diff-notice" lang="en"><div class="mw-diff-empty">(No difference)</div> </td></tr></table>

imported>Virgilio https://wiki.xenbase.org/xenwiki/index.php?title=CSF_extract,_sara%27s_protocol_(Stukenberg_lab)&diff=47232&oldid=prev 2011-11-14T15:08:19Z

<p>Created page with &quot;Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&amp;personId=1311&amp;tabId=0] link to protocol page [http://people.v...&quot;</p> <p><b>New page</b></p><div>Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&amp;personId=1311&amp;tabId=0]<br /> <br /> <br /> link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]<br /> <br /> <br /> <br /> Sara&#039;s CSF EXTRACT<br /> <br /> Preparation:<br /> <br /> 2-3 L of 1X MMR, make night before, store at 20°C.<br /> <br /> 200mL de-jelly solution, 2% cysteine, pH to 7.8 with 0.9mL 10N NaOH.<br /> <br /> 750mL XB:<br /> 10mM K-HEPES<br /> 50mM sucrose<br /> 1X XB<br /> 11uL 10N KOH per 100mL to keep pH 7.7<br /> <br /> 250mL CSF-XB:<br /> 250mL XB (from above)<br /> 5mM EGTA<br /> 1mM MgCl2 <br /> <br /> 100mL CSF-XB + PIs:<br /> 100mL CSF-XB (from above)<br /> 10 ug/mL LPC<br /> <br /> 2M Sucrose<br /> <br /> LPC<br /> <br /> Cytochalasin<br /> <br /> 40X Energy Mix<br /> <br /> Sperm<br /> <br /> Extract Fix: <br /> 60% Glycerol<br /> 1X MMR<br /> 1ug/mL Hoescht<br /> 10% Formaldehyde<br /> <br /> Sperm Dilution Buffer (5X)<br /> <br /> 10X Calcium: 4mM CaCl2 in 1X Sperm Dilution Buffer<br /> <br /> If the extracts will be spun onto coverslips, then also need:<br /> <br /> Fixation Buffer:<br /> BRB80<br /> 20% Glycerol<br /> 0.5% TX-100<br /> 4% Formaldehyde<br /> <br /> Cushion:<br /> BRB80<br /> 30% Glycerol<br /> <br /> -20°C MeOH<br /> <br /> <br /> <br /> Making a CSF Extract<br /> <br /> Collect eggs.<br /> <br /> Wash eggs in MMR 2-3X.<br /> <br /> Remove as much MMR as possible, and pour on dejellying solution.<br /> <br /> De-jelly 7-10 min. Rinse 1X in de-jelly. While eggs are de-jellying, add 1mL CSF-XB +PIs to centrifuge tubes. Add 10uL 10mg/mL cytochalasin and flick tube well.<br /> <br /> Wash de-jellied eggs 3-4X in XB. <br /> <br /> Wash eggs 2-3 X in CSF-XB.<br /> <br /> Wash eggs 2X in CSF-XB + PIs. Leave in small volume of CSF-XB+PIs.<br /> <br /> Gently pipet eggs into centrifuge tubes, put pipet below meniscus.<br /> <br /> Remove excess buffer.<br /> <br /> Spin 10 sec at 1500 rpm (setting 4) in clinical centrifuge/<br /> <br /> Aspirate off buffer. Add 100uL Versilube to top<br /> <br /> Pack eggs: spin at 2000rpm (setting 5) for 30 sec and full speed for 1min in clinical centrifuge.<br /> <br /> Aspirate off versilube and buffer from top of eggs.<br /> <br /> Centrifuge packed eggs at 10,000 rpm for 15 min at 16°C in the SW55.1 rotor.<br /> <br /> Collect cytoplasmic layer of extract with 18-guage needle into a 1-cc syringe. Avoid bubbles. <br /> <br /> Transfer to new tube and estimate volume.<br /> <br /> Add 1/1000 volume LPC.<br /> <br /> Add 1/1000 volume cytochalasin.<br /> <br /> 1/40 volume 40X energy mix.<br /> <br /> Add 1/40 volume 2M sucrose.</div>

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