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<p class="MsoNormal" style="line-height:-5%"><span class="heading1"><span style="font-size:11.0pt;line-height:-5%;font-family:"Arial",sans-serif"><o:p> </o:p></span></span></p>
<p class="MsoNormal"><span class="heading1"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Our collective thanks to Gert Veenstra for this protocol!</span></span><span class="heading1"><span style="font-weight:normal;font-style:normal"><o:p></o:p></span></span></p>
<p class="MsoNormal"><span class="heading1"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></span></p>
<p class="MsoNormal"><span class="heading1"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></span></p>
<p class="MsoNormal"><span class="heading1"><span style="font-size:16.5pt;font-family:"Arial",sans-serif">Multi-purpose embryo extracts (a freon-free protocol)</span></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal" style="line-height:-5%"><span style="font-size:11.0pt;line-height:-5%;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Diluted yolk-depleted embryo extracts: Yolk can be depleted from whole-embryo lysates by simple centrifugation.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Please note that high salt or ionic detergents (SDS) partially solubilize yolk, which interferes with clearing the extract.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Procedure<o:p></o:p></span></b></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<ul style="margin-top:0in" type="disc">
<li class="MsoNormal" style="line-height:150%;mso-list:l2 level1 lfo3"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif">Collect staged embryos (up to 50 / tube)<o:p></o:p></span></li><li class="MsoNormal" style="line-height:150%;mso-list:l2 level1 lfo3"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif">Remove excess buffer / water<o:p></o:p></span></li><li class="MsoNormal" style="line-height:150%;mso-list:l2 level1 lfo3"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif">Snap freeze in liquid nitrogen / dry ice<o:p></o:p></span></li><li class="MsoNormal" style="line-height:150%;mso-list:l2 level1 lfo3"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif">Store at -80°C<o:p></o:p></span></li></ul>
<p class="MsoNormal" style="line-height:150%"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<ul style="margin-top:0in" type="disc">
<li class="MsoNormal" style="line-height:150%;mso-list:l2 level1 lfo3"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif">Add 4 volume equivalents of room temperature WCE-LS buffer to frozen embryos<o:p></o:p></span></li></ul>
<p class="MsoNormal" style="line-height:150%"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif"> NB One embryo corresponds to approx. 1 μl<o:p></o:p></span></p>
<p class="MsoNormal" style="line-height:150%"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif"> Final concentration 0.2 embryo equivalent / μl (<i>X. laevis</i>)<o:p></o:p></span></p>
<ul style="margin-top:0in" type="disc">
<li class="MsoNormal" style="line-height:150%;mso-list:l3 level1 lfo7"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif">Quick-thaw and homogenize embryos by pipetting up and down (yellow tip), then keep on ice<o:p></o:p></span></li><li class="MsoNormal" style="line-height:150%;mso-list:l3 level1 lfo7"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif">Spin in eppendorf centrifuge for 5 mins at max speed at 4ºC<o:p></o:p></span></li><li class="MsoNormal" style="line-height:150%;mso-list:l3 level1 lfo7"><span style="font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif">Quick-freeze (aliquots of) supernatant in liquid nitrogen / dry ice, store at -80°C<o:p></o:p></span></li></ul>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Low-salt whole cell extract (WCE-LS) buffer</span></b><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"> 20 mM Tris pH 7.5<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"> 70 mM KCl<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"> 1 mM EDTA<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"> 10 % Glycerol<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"> 5 mM DTT<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"> 0.125 % Nonidet P40 / IGEPAL CA-630<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Add “Complete” protease inhibitor, or cocktail of leupeptin, pepstatin A aprotinin 1 μg/μl each<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Example:</span></b><span style="font-size:11.0pt;font-family:"Arial",sans-serif">
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Veenstra GJ, Destrée OH, Wolffe AP.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Translation of maternal TATA-binding protein mRNA potentiates basal but not activated transcription in Xenopus embryos at the midblastula transition.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Mol Cell Biol. 1999 Dec;19(12):7972-82.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">PMID: 10567523; PMCID: PMC84882.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">doi:10.1128/MCB.19.12.7972.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Arial",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
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