Archived Protocol

<!doctype html public "-//w3c//dtd html 4.0 transitional//en"> <html> <head> <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1"> <meta name="Description" content="Cell and Xenopus methods ala Klymkowsky et al"> <meta name="description" content="MCD Biology 1150"> <meta name="keywords" content="Cell biology, Evolution, Disease"> <meta name="GENERATOR" content="Mozilla/4.7 (Macintosh; I; PPC) [Netscape]"> <meta name="KeyWords" content="Cell Biology, Xenopus"> <meta name="Content-Type" content="text/html; charset=iso-8859-1"> <title>Klymkowsky Lab Methods</title> </head> <style type="text/css"> </style> <body text="#000000" bgcolor="#FFFFFF" link="#000099" vlink="#990000" alink="#990066"> <table width="600" border="0" cellpadding="0"> <tr> <td width="341"><div align="left"> <table width="99%" border="0"> <tr> <td width="49%"><img src=http://spot.colorado.edu/~klym/Graphics/Manual.jpg width="175" height="160"></td> <td width="51%"><div align="left"><font color="#000000" face="Book Antiqua"><i><font size="+1">Xenopus oocyte isolation, maturation, enucleation, fractionation & injection.</font></i></font></div></td> </tr> </table> </div> <div align="right"> <table width="100%" border="0"> <tr> <td width="66%"><font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua"><strong><font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua"><strong><font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua"><strong><font face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/%22http://spot.colorado.edu/~klym/methods.htm " target="_self"><img src=http://spot.colorado.edu/~klym/Graphics/ArrowLEFT.GIF width="14" height="14"></a></font></strong></font></em></font></strong></font></em></font><font face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/%22http://spot.colorado.edu/~klym/methods.htm " target="_self"> Table of Methods</a><a name="top"></a></font></strong></font></em></font></td> <td width="34%"><div align="right"><font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua">Oocytes stained for nuclear lamins</font></em></font> </div> <div align="right"><strong></strong></div></td> </tr> </table> </div></td> <td width="245"> <div align="right"><img src=http://spot.colorado.edu/~klym/Graphics/lamina.GIF width="244" height="226"></div></td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <hr> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td width="148"><strong><font size="-1" face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/~klym/Methods/oocyteIsolation.htm#maturation>oocyte maturation</a></font></strong></td> <td width="138"><strong><font size="-1" face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/~klym/Methods/oocyteIsolation.htm#enucleation>enucleation</a> </font></strong></td> <td width="138"><font size="-1" face="Arial, Helvetica, sans-serif"><strong><a href=http://spot.colorado.edu/~klym/Methods/oocyteIsolation.htm#fractionation>fractionation</a></strong></font></td> <td width="166"><font size="-1" face="Arial, Helvetica, sans-serif"><strong><a href=http://spot.colorado.edu/~klym/Methods/oocyteIsolation.htm#inject>injection</a></strong></font></td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <hr> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <strong><font face="Arial, Helvetica, sans-serif">oocyte isolation </font></strong> </td> </tr> </table> <strong></strong> <table width="600" border="0"> <tr> <td><ol> <li><font face="Arial, Helvetica, sans-serif">Anethesize female using benzocaine - put ~5ml 6% benzocaine (stock solution in 100% ethanol) into 1L water.<br> - leave animal until it is limp and completely unresponsive.<br> - place animal onto paper towel</font></li> <li><font face="Arial, Helvetica, sans-serif">Remove ovaries (both) and wash with Ca2+, Mg2+ free Ringers</font></li> <li><font face="Arial, Helvetica, sans-serif">Dissociate overnight at 16°C in 0.1% collagenase in 5mg/ml ovalbumin in Ca2+/Mg2+-free Ringers supplemented with 10mM NaPO4 .Use rocker. To dissociate a complete ovary, use 20m total volume. </font></li> <li> <div align="right"><font face="Arial, Helvetica, sans-serif">Recover oocytes and wash twice in OR2 (which can be supplemented with 5% dialyzed calf serum for long term culture) Media should supplemented with antibiotics - either gentamycin (50µg/ml) or penicillin/streptomycin (from 100X stock).</font><font face="Arial, Helvetica, sans-serif"><br> </font> <table width="100%" border="0"> <tr> <td><font face="Arial, Helvetica, sans-serif"><strong>Modified Ringers Solution (MRS) </strong><br> 110mM NaCl<br> 2mM KCl<br> 1mM MgCl2 <br> 2mM CaCl2<br> 2mM NaHCO3<br> 5mM HEPES pH 7.8 </font></td> <td><p><font face="Arial, Helvetica, sans-serif"><strong>OR2 media:</strong><br> 82.5mM NaCl<br> 2.5mM KCl<br> 1mM CaCl2<br> 1mM MgCl2<br> 1mM Na2HPO4<br> 5mM HEPES pH 7.8 </font></p></td> </tr> </table> <font face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/~klym/Methods/oocyteIsolation.htm#top><font size="-1">back to the top</font></a></font></div> </li> </ol></td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <font face="Arial, Helvetica, sans-serif"><a name="maturation"></a></font> <hr> </td> </tr> </table> <div align="left"><font color="#000000" face="Book Antiqua"><b></b></font></div> <table width="600" border="0" cellpadding="0"> <tr> <td> <p><font face="Arial, Helvetica, sans-serif"><strong>Oocyte maturation</strong>:</font></p> <p><font face="Arial, Helvetica, sans-serif"><br> Make oocyte media 5µg/ml progesterone (progesterone made as 5mg/ml stock in 100% ethanol). </font></p> <p><font face="Arial, Helvetica, sans-serif">Animal pole pigment clearing should be apparent by 3 to 4 hours, but may take as long as 6 to 8 hours to become evidence. </font></p> <p><font face="Arial, Helvetica, sans-serif">Even in cases where animal pole spot does not appear, the oocytes can have entered M-phase, as judged by disappearance of nucleus, breakdown of keratin filaments.</font></p> <p align="right"><font size="-1" face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/~klym/Methods/oocyteIsolation.htm#top>top of this page</a></font><font face="Arial, Helvetica, sans-serif"> </font></p> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <font face="Arial, Helvetica, sans-serif"><strong> </strong><a name="enucleation"></a></font> <hr> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <p><font face="Arial, Helvetica, sans-serif"><strong>Oocyte enucleation</strong><br> </font></p> <p><font face="Arial, Helvetica, sans-serif"> transfer oocytes into MRS<br> </font></p> <p><font face="Arial, Helvetica, sans-serif"> use a pair of blunt forceps, gently hold oocyte and puncture with a unblunted 26/27 gauge needle.<br> </font></p> <p><font face="Arial, Helvetica, sans-serif">gently squeeze oocyte until germinal vesicle appears. you can use the needle to tease GV out. use only enucleated egg in which GV is removed untorn.<br> </font></p> <p><font face="Arial, Helvetica, sans-serif"><strong>note</strong>:usually the GV remains fairly round throughout the enucleation process, but sometimes it is almost stringy. </font></p> <p><font face="Arial, Helvetica, sans-serif">I would not use such oocytes. <br> </font></p> <p><font face="Arial, Helvetica, sans-serif"> place enucleated oocytes into MRS - allow to heal for 30 to 60 minutes. even some very ugly looking oocytes will heal completely under these conditions!</font></p> <p> </p> <p align="right"><font size="-1" face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/~klym/Methods/oocyteIsolation.htm#top>top of this page</a></font><font face="Arial, Helvetica, sans-serif"> </font> </p> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <font face="Arial, Helvetica, sans-serif"><strong><a name="fractionation"></a></strong></font> <hr> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <p><font face="Arial, Helvetica, sans-serif"><strong>Preparation of insoluble and soluble fractions from oocytes</strong></font></p> <p><font face="Arial, Helvetica, sans-serif"><br> Eggs and embryos must be dejellied with 2% cysteine pH 8.0 / wash 3x with Ringers.<br> </font></p> <p><font face="Arial, Helvetica, sans-serif">Place oocytes, eggs or embryos into microfuge tube & remove excess liquid. </font></p> <p><font face="Arial, Helvetica, sans-serif">T</font><font face="Arial, Helvetica, sans-serif">ypical experiment uses 5 to 20 oocyte/embryos per gel lane. <br> wash once in MSB buffer<br> </font></p> <table width="100%" border="0"> <tr> <td width="46%"><p><font face="Arial, Helvetica, sans-serif"><strong>Medium salt extraction buffer</strong></font></p> <p><font face="Arial, Helvetica, sans-serif"><br> 150mM NaCl <br> 50mM NaF<br> 10mM EDTA<br> 10mM Tris-base pH 7.4 </font></p></td> <td width="54%"><font face="Arial, Helvetica, sans-serif"><strong>XEX buffer:</strong> <br> 1.5M KCl <br> 0.3M sucrose <br> 50mM NaF<br> 10mM EDTA.<br> 10mM Tris pH 7.4 <br> 0.5% NP40</font></td> </tr> </table> <p><font face="Arial, Helvetica, sans-serif">homogenize first is MSB using 1.5ml in a 1.7ml microfuge tube.<br> carefully pipette up and down with pasteur pipette. be careful not to splash stuff out of the microfuge tube-it is easy to do!. - <br> </font></p> <p><font face="Arial, Helvetica, sans-serif">centrifuge in microfuge for 15 minutes (at 4°C).<br> </font></p> <p><font face="Arial, Helvetica, sans-serif">aspirate supernatant from the top of the tube <br> </font></p> <p><font face="Arial, Helvetica, sans-serif">take care to remove yolk from the top of the "pellet" microfuge tube <br> </font></p> <p><font face="Arial, Helvetica, sans-serif">re-suspend pellet in 1.5ml XEX <br> </font></p> <p><font face="Arial, Helvetica, sans-serif">centrifuge resuspended pellet for 15 minutes at 4°C.<br> </font></p> <p><font face="Arial, Helvetica, sans-serif">solubilize recovered pellet in either SDS-page or IEF sample buffer. </font></p> <p align="right"><font size="-1" face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/~klym/Methods/oocyteIsolation.htm#top>top of this page</a></font><font face="Arial, Helvetica, sans-serif"><strong></strong></font> </p> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <hr> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <strong><font face="Arial, Helvetica, sans-serif">Oocyte injection - coming <a name="inject"></a></font></strong></td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td> <hr> </td> </tr> </table> <table width="600" border="0" cellpadding="0"> <tr> <td width="197"> <font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua"><strong><font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua"><strong><font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua"><strong><font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua"><strong><font size="-1" face="Arial, Helvetica, sans-serif"><em><font face="Book Antiqua"><strong><font face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/%22http://spot.colorado.edu/~klym/methods.htm " target="_self"><img src=http://spot.colorado.edu/~klym/Graphics/ArrowLEFT.GIF width="14" height="14"></a></font></strong></font></em></font></strong></font></em></font></strong></font></em></font><font face="Arial, Helvetica, sans-serif"></font></strong></font></em></font><font face="Arial, Helvetica, sans-serif"><a href=http://spot.colorado.edu/%22http://spot.colorado.edu/~klym/methods.htm " target="_self"> Table of Methods</a></font></strong></font></em></font></td> <td width="150">  </td> <td width="245"><div align="right"><font size="-1" face="Arial, Helvetica, sans-serif">revised 3 August 2002</font></div></td> </tr> </table> <div align="left"> </div> <div align="left"> </div> <div align="left"> </div> <div align="left"> </div> <p> <p><font color="#000000" face="Book Antiqua"><br>   </font> <div align="left"></div> </body> </html>