Edinburgh/Notebook/Gels

From 2008.igem.org

(Difference between revisions)
(New page: <html> <head> <title>Edinburgh iGem 2008</title> <script type="text/javascript"> //Drop Down Tabs Menu- Author: Dynamic Drive (http://www.dynamicdrive.com) //Created: May 16th, 07' var ta...)
 
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<html>
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<div id="header">{{Template:Team:Edinburgh/Templates/Header}}</div>
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<head>
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<title>Edinburgh iGem 2008</title>
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<script type="text/javascript">
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//Drop Down Tabs Menu- Author: Dynamic Drive (http://www.dynamicdrive.com)
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//Created: May 16th, 07'
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var tabdropdown={
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'''[[Team:Edinburgh/Notebook|< Back to Notebook]]'''
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* disappeardelay: 200, //set delay in miliseconds before menu disappears onmouseout
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disablemenuclick: false, //when user clicks on a menu item with a drop down menu, disable menu item's link?
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enableiframeshim: 1, //1 or 0, for true or false
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//No need to edit beyond here////////////////////////
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dropmenuobj: null, ie: document.all, firefox: document.getElementById&&!document.all, previousmenuitem:null,
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currentpageurl: window.location.href.replace("http://"+window.location.hostname, "").replace(/^\//, ""), //get current page url (minus hostname, ie: http://www.dynamicdrive.com/)
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if (this.ie || this.firefox)
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this.dropmenuobj.onmouseout=function(e){tabdropdown.dynamichide(e, obj)}
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this.dropmenuobj.style.top=this.dropmenuobj.y-this.clearbrowseredge(obj, "bottomedge")+obj.offsetHeight+1+"px"
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this.previousmenuitem=obj //remember main menu item mouse moved out from (and into current menu item)
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dynamichide:function(e, obj2){ //obj2 refers to tab menu item mouse is currently over
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var evtobj=window.event? window.event : e
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if (this.ie&&!this.dropmenuobj.contains(evtobj.toElement))
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this.delayhidemenu(obj2)
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this.delayhidemenu(obj2)
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this.delayhide=setTimeout(function(){tabdropdown.dropmenuobj.style.visibility='hidden'; if (obj2.parentNode.className.indexOf('default')==-1) obj2.parentNode.className=''},this.disappeardelay) //hide menu
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clearhidemenu:function(){
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if (this.delayhide!="undefined")
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clearTimeout(this.delayhide)
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this.shimobject.style.left=this.dropmenuobj.style.left
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this.shimobject.style.top=this.dropmenuobj.style.top
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this.shimobject.style.display=(this.dropmenuobj.style.visibility=="visible")? "block" : "none"
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}
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},
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hideshim:function(){
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if (this.enableiframeshim && typeof this.shimobject!="undefined")
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this.shimobject.style.display='none'
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},
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isSelected:function(menuurl){
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var menuurl=menuurl.replace("http://"+menuurl.hostname, "").replace(/^\//, "")
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return (tabdropdown.currentpageurl==menuurl)
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},
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init:function(menuid, dselected){
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this.standardbody=(document.compatMode=="CSS1Compat")? document.documentElement : document.body //create reference to common "body" across doctypes
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var menuitems=document.getElementById(menuid).getElementsByTagName("a")
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for (var i=0; i<menuitems.length; i++){
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var relvalue=menuitems[i].getAttribute("rel")
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document.getElementById(relvalue).firstlink=document.getElementById(relvalue).getElementsByTagName("a")[0]
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menuitems[i].onmouseover=function(e){
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var event=typeof e!="undefined"? e : window.event
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tabdropdown.dropit(this, event, this.getAttribute("rel"))
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}
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if (dselected=="auto" && typeof setalready=="undefined" && this.isSelected(menuitems[i].href)){
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menuitems[i].parentNode.className+=" selected default"
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var setalready=true
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else if (parseInt(dselected)==i)
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menuitems[i].parentNode.className+=" selected default"
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}
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}
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</script>
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<style>
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.ddcolortabs{
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padding: 0;
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width: 100%;
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background: transparent;
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voice-family: "\"}\"";
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voice-family: inherit;
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}
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.ddcolortabs ul{
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font: normal 11px Arial, Verdana, sans-serif;
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margin:0;
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padding:0;
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list-style:none;
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}
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.ddcolortabs li{
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display:inline;
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margin:0 2px 0 0;
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padding:0;
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text-transform:uppercase;
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}
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+
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.ddcolortabs a{
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float:left;
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color: white;
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background: black url(media/color_tabs_left.gif) no-repeat left top;
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margin:0 2px 0 0;
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padding:0 0 1px 3px;
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text-decoration:none;
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letter-spacing: 1px;
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}
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.ddcolortabs a span{
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float:left;
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display:block;
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background: transparent url(media/color_tabs_right.gif) no-repeat right top;
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padding: 4px 8px 2px 7px;
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}
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.ddcolortabs a span{
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float:none;
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.ddcolortabs a:hover{
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background-color: #591f20;
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}
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.ddcolortabs a:hover span{
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background-color: #591f20;
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}
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.ddcolortabs .selected a, #ddcolortabs .selected a span{ /*currently selected tab*/
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background-color: #591f20;
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}
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.ddcolortabsline{
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clear: both;
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padding: 0;
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width: 100%;
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height: 8px;
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line-height: 8px;
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background: black;
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border-top: 1px solid #fff; /*Remove this to remove border between bar and tabs*/
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-
}
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-
 
+
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/* ######### Style for Drop Down Menu ######### */
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-
 
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.dropmenudiv_a{
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position:absolute;
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top: 0;
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border: 1px solid black; /*THEME CHANGE HERE*/
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border-top-width: 8px; /*Top border width. Should match height of .ddcolortabsline above*/
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-
border-bottom-width: 0;
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-
font:normal 12px Arial;
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-
line-height:18px;
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-
z-index:100;
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-
background-color: white;
+
-
width: 200px;
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-
visibility: hidden;
+
-
}
+
-
 
+
-
 
+
-
.dropmenudiv_a a{
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-
width: auto;
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-
display: block;
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-
text-indent: 5px;
+
-
border-top: 0 solid #678b3f;
+
-
border-bottom: 1px solid #678b3f; /*THEME CHANGE HERE*/
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-
padding: 2px 0;
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-
text-decoration: none;
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-
color: black;
+
-
}
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-
 
+
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* html .dropmenudiv_a a{ /*IE only hack*/
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-
width: 100%;
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-
}
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-
 
+
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.dropmenudiv_a a:hover{ /*THEME CHANGE HERE*/
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-
background-color: #8a3c3d;
+
-
color: white;
+
-
}
+
-
</style>
+
-
</head>
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-
 
+
-
<!-- CSS for Drop Down Tabs Menu #1 -->
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<link rel="stylesheet" type="text/css" href="ddcolortabs.css" />
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-
 
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<div id="colortab" class="ddcolortabs">
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<ul>
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<li><a href="https://2008.igem.org/Team:Edinburgh" title="Home"><span>Home</span></a></li>
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-
<li><a href="https://2008.igem.org/Team:Edinburgh/Project" title="Project" rel="dropmenu1_a"><span>The Project</span></a></li>
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<li><a href="https://2008.igem.org/Team:Edinburgh/Team" title="Team" ><span>The Team</span></a></li>
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<li><a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Edinburgh" title="Project" rel="dropmenu1_a"><span>BioBrick Parts</span></a></li>
+
-
<li><a href="https://2008.igem.org/Team:Edinburgh/Modeling" title="Modelling" rel="dropmenu2_a"><span>Modelling</span></a></li>
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<li><a href="https://2008.igem.org/Team:Edinburgh/Notebook" title="Notebook"><span>Notebook</span></a></li>
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-
</ul>
+
-
</div>
+
-
<div class="ddcolortabsline">&nbsp;</div>
+
-
<div id="dropmenu1_a" class="dropmenudiv_a">
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<a href="https://2008.igem.org/Team:Edinburgh/Team">Overview</a>
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<a href="https://2008.igem.org/Team:Edinburgh/Team">Step1</a>
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<a href="https://2008.igem.org/Team:Edinburgh/Team">Step2</a>
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</div>
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<script type="text/javascript">
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//SYNTAX: tabdropdown.init("menu_id", [integer OR "auto"])
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tabdropdown.init("colortab", 3)
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</script>
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</html>
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 +
== Gels ==
'''Gel 1:''' PCR reactions P1 (''dxs''), P2 (''appY''), P3 (''glgC''). (25.06.08)<br />
'''Gel 1:''' PCR reactions P1 (''dxs''), P2 (''appY''), P3 (''glgC''). (25.06.08)<br />
'''Gel 2:''' EcoRI/PstI digests of M1 to M6 (pSB1A2+''dxs''). (30.06.08)<br />
'''Gel 2:''' EcoRI/PstI digests of M1 to M6 (pSB1A2+''dxs''). (30.06.08)<br />
-
*Gel 3:* minipreps M2, M3, M4, M6, EcoRI, plus L1 and L2
+
'''Gel 3:''' minipreps M2, M3, M4, M6, EcoRI, plus L1 and L2. (01.07.08)<br />
-
*Gel 4:* minipreps M7 to M12, undigested
+
'''Gel 4:''' minipreps M7 (BABEL1+''glgC'') and M8 to M12 (BABEL2+''glgC''), undigested. (01.07.08)<br />
-
*Gel 5:* minipreps M7 to M12, EcoRI/PstI
+
'''Gel 5:''' minipreps M7 to M12, EcoRI/PstI. (01.07.08)<br />
-
*Gel 6:* minipreps M2, M3, M4, M6, HindIII; also P4 and P5 (fusion PCR)
+
'''Gel 6:''' minipreps M2, M3, M4, M6, HindIII; also P4 (BABEL1+''glgC'' fusion PCR)and P5 (BABEL2+''glgC'' fusion PCR). (01.07.08)<br />
-
*Gel 7:* minipreps M10 and M11, EcoRI (two versions of this gel)
+
'''Gel 7:''' minipreps M10 and M11, EcoRI (two versions of this gel). (04.07.08)<br />
-
*Gel 8:* PCR products P6 and P7 (mutagenic PCR on M11)
+
'''Gel 8:''' PCR products P6 and P7 (mutagenic PCR on ''glgC'' [M11]). (04.07.08)<br />
-
*Gel 9:* maxipreps X1 and X2, etc.
+
'''Gel 9:''' digests of maxipreps X1 (J33201) and X2 (pSB1A2+''dxs''). (08.07.08)<br />
-
*Gel 10:* minipreps M13 to M18, E/P digests.
+
'''Gel 10:''' minipreps M13 to M18, EcoRI/PstI digests. (09.07.08)<br />
-
*Gel 11:* minipreps M19 to M24, EcoRI digests, plus PCR P11.
+
'''Gel 11:''' M19 to M24 (BABEL2+''glgC''-mut2) EcoRI digests, plus PCR P11 (rbs+''dxs''). (10.07.08)<br />
-
*Gel 12:* minipreps M25 to M30 of possible rbs+dxs clones: none look right.
+
'''Gel 12:''' SacI/SpeI digests of M25 to M30 (possible rbs+''dxs'' clones): none look right. Also shown is P12 (rbs+''dxs'' fusion PCR product). (13.07.08)<br />
-
*Gel 13:* PCR P13 and P14 to mutated second EcoRI site on glgC clones M19 and M22.
+
'''Gel 13:''' PCR P13 and P14 to mutate out first EcoRI site from BABEL2+''glgC''-mut2 clones M19 and M22. (14.07.08)<br />
-
*Gel 14:* minipreps M31 to M42 (crtB and crtI clones) E/P, and P15 (rbs+appY)
+
'''Gel 14:''' minipreps M31 to M42 (pSB1A2+''crtB'' and pSB1A2+''crtI'' clones) EcoRI/PstI digests, and P15 (rbs+''appY'') (15.07.08)<br />
-
*Gel 15:* redigest of M32, M36 (crtB), M39, M40 (crtI) E/P; unsuccessful.
+
'''Gel 15:''' redigest of M32, M36 (pSB1A2+''crtB''), M39, M40 (pSB1A2+''crtI'') with EcoRI/PstI; unsuccessful. (17.07.08)<br />
-
*Gel 16:* purified PCR products for cenA, cenB, cenC, cex (P16, 17, 18, 19); unsuccessful.
+
'''Gel 16:''' purified PCR products for ''cenA'', ''cenB'', ''cenC'', ''cex'' (P16 to P19); unsuccessful. (18.07.08)<br />
-
*Gel 17:* PCR products for cenA, cenB, cenC, cex (P20, 21, 22, 23) ~ second attempt; unsuccessful.
+
'''Gel 17:''' PCR products for ''cenA'', ''cenB'', ''cenC'', ''cex'' (P20 to P23); unsuccessful. (18.07.08)<br />
-
*Gel 21:* P26 (rrnB from C fimi); unsuccessful.
+
'''Gel 18:''' EcoRI/PstI digests of M43 to M47 (BABEL2+''glgC''-mut1,2) and M48 (pSB1A2+rbs+''dxs''). M43 to M47 all look about right. M48 is ambiguous, could be incomplete deigestion, need to repeat. (19.07.08: CF)<br />
-
*Gel 22:* P27 (crtY ~ unsuccessful), P28, P29 (crtB, crtI ~ successful).
+
'''Gel 19:''' P24 (''cenA''), P25 (''cex'') as well as SacI/SpeI digest of M48 (pSB1A2+rbs+''dxs'') and single (EcoRI) and double (EcoRI/PstI) digests of M36 (pSB1A2+''crtB'') and M42 (pSB1A2+''crtI''). (20.07.08: CF)<br />
-
*Gel 23:* P30 (repeat rrnB from C fimi) and PZNTA. Successful.
+
'''Gel 20:''' L15 (rbs+''dxs'' with Edinbrick1) and L16 (rbs+''appY'' with Edinbrick1) both failed. (22.07.08: OG, Yan)<br />
-
*Gel 25:* P36 (glgC excision ~ successful), P37 (crtY ~ failed).
+
'''Gel 21:''' P26 (''rrnB'' from ''C. fimi''); unsuccessful. (22.07.08: AM)<br />
-
*Gel 26:* P38~43 (cenA, cenB, cenC, cex from heat-killed cell suspension; cenA and cenB from 'impure' DNA solution.
+
'''Gel 22:''' P27 (''crtY'', unsuccessful), P28 (pSB1A2+rbs+''crtB'', successful) and P29 (pSB1A2+rbs+''crtI'', successful). (23.07.08: AM, Yan, OG)<br />
-
*Gel 27:* Repeat of Gel 26 but stained with SYBRsafe; putative cenA and cex fragments cut out.
+
'''Gel 23:''' P30 (repeat ''rrnB'' from ''C. fimi'') and pZntA, both successful. (23.07.08: AM)<br />
-
*Gel 28:* M49~54 (pSB1A2-rbs+crtI) digested with EcoRI.
+
'''Gel 24:''' Shows failure of P32-P35 (''cenA'', ''cenB'', ''cenC'' and ''cex''). (24.07.08: AM)<br />
-
*Gel 29:* M55~60 (PZntA), M61~66 (CrtE+RBS) all digested with EcoRI and PstI.
+
'''Gel 25:''' P36 (''glgC''-mut1,2 excision from BABEL2, successful), P37 (''crtY'', failed). (25.07.08: AH)<br />
-
*Gel 30:* P46 (cenA), P47 (cex) from heat killed cell soln, annealing 65C. Failed. (30/07/08)
+
'''Gel 26:''' P38-41 (cenA, cenB, cenC, cex from heat-killed cell suspension) and P42-43 (cenA and cenB from 'impure' DNA solution). (28.07.08: AM)<br />
-
*Gel 31*: M49 (X,SX,sac/spt), M50(X,SX,sac/spt), M63(E,EP), M67(E/P, sac1/spe1), P38(_cenA_). (30/07/08: OG)
+
'''Gel 27:''' Repeat of Gel 26 but stained with SYBRsafe. Putative cenA and cex fragments cut out. (29.07.08: AM)<br />
-
*Gel 32:* M55 to M60 double digestion with EcorI and PstI, and single digestion with EcoRI only. (30/07/08: Yan)
+
'''Gel 28:''' M49-54 (pSB1A2+rbs+''crtI'') digested with EcoRI. (29.07.08: CF)<br />
-
*Gel 33:* M68~71 (pSB1A2-glgC m1+m2) digested with EcoRI+PstI, M72~75 (pSB1A2-rbs+dxs) digested with EcoRI, M76~79 (Babel2\+_crtE_) digested with EcoRI+PstI. See 01.08.2008 entry for results.
+
'''Gel 29:''' M55-60 (pSB1A2+pZntA), M61-66 (pSB1A2+rbs+''CrtE'') digested with EcoRI/PstI. Shows that ligations L23 and L24 (respectively) failed. (29.07.08: Yan, AM)<br />
-
*Gel 34:* M68~69 (pSB1A2-glgC m1+m2) digested with EcoRI alone and EcoRI+PstI, M72 (pSB1A2-rbs+dxs) digested with EcoRI alone and SacI+SpeI. See 01.08.2008 entry for results.
+
'''Gel 30:''' P46 (''cenA''), P47 (''cex'') from heat killed cell solution, annealing 65C. Gel shows failure of the PCR. (30/07/08)<br />
-
*Gel 35:* P36 and L25. P36 successful, L25 yields clear glgC-length band but faint vector-length band.
+
'''Gel 31:''' P48 (''crtY'') and digests of M49 and M50 (pSB1A2-rbs+''crtI'') with a) XbaI, b) SpeI/XbaI, c) sac/speI, M63 (BABEL2+rbs+''crtE'') with a) EcoRI, b) EcoRI/PstI, M67 (pSB1A2+rbs+''crtB'') with a) EcoRI/PstI, b) sacI/speI. (30/07/08: OG)<br />
-
*Gel 36:* Double digestion of M80 to M96 with EcoRI and PstI, and PCR production of third mutagenesis of glgc mutant 1&2. Results were good for M80-82 (rbs\-_appY_\-pSB1A2), M84-85 (rbs\-_crtB_\-pSB1A2), M92 (_cex_\-pSB1A2) and P50 (_glgC_\-mut1,2+3). Other results were poor. (YAN&OG).
+
'''Gel 32:''' M55-M60 (pSB1A2+pZntA) double digestion with EcoRI/PstI, and single digestion with EcoRI. (30.07.08: Yan)<br />
-
*Gel 37:* Double digestion of M97 to M108 with EcoRI and PstI.
+
'''Gel 33:''' M68-71 (pSB1A2+''glgC''-mut1,2) digested with EcoRI/PstI, M72-75 (pSB1A2+rbs+''dxs'') digested with EcoRI, M76-79 (BABEL2+''crtE'') digested with EcoRI/PstI. (01.08.2008: AM)<br />
-
*Gel 39:* Analytical digests using a) EcoRI and b) EcoRI/PstI of M70, M71 (both pSB1A2\-_glgC_ mut1+2) and M92 (pSB1A2\-_cex_). Results do not look good. Bands are wrong size to correspond to vector and no bands match an expected insert size. (06/08/08: CF)
+
'''Gel 34:''' M68-69 (pSB1A2+''glgC''-mut1,2) digested with EcoRI alone and EcoRI+PstI, M72 (pSB1A2+rbs+''dxs'') digested with EcoRI alone and SacI/SpeI. (01.08.2008: AM)<br />
-
*Gel 40* PCR products of PcstA (P54) (and SOB). Results look promising (fairly prominent band <500bp). (07.08.08: CF)
+
'''Gel 35:''' P36 (''glgC''-mut1,2) and L25 (pSB1A2+''glgC''-mut1,2). P36 successful, L25 yields clear ''glgC''-length band but faint vector-length band. (01.08.2008: AM)<br />
-
*Gel 41:* PCR products of P55 to P58 (11.08.08: YAN)
+
'''Gel 36:''' P50 (''glgC''-mut1,2,3) and double digestion of M80-M96 with EcoRI/PstI. Results were good for M80-M82 (pSB1A2+rbs+''appY''), M84-M85 (pSB1A2+rbs+''crtB''), M92 (pSB1A2+''cex'') and P50. Other results were poor. (04.08.08: Yan, OG).<br />
-
*Gel 42:* _C. fimi_ genomic DNA and PCR products P61~P63 from _C. fimi_ genomic DNA (11.08.08: CF)
+
'''Gel 37:''' Double digestion of M97-M104 (pSB1A2+''cenA'') and M105-M108 (pSB1A2+''cex'') with EcoRI/PstI. Results were very poor apart from M97. (05.08.08: Yan)<br />
-
*Gel 43:* Double digests of BABEL2\-_crtE_ (M79) and Edinbrick1 with EcoRI and PstI, showing a single band at \~3kb for BABEL2\+_crtE_ and bands at \~2.5kb and 800bp for Edinbrick1. (11.08.08: AH)
+
'''Gel 38:''' Re-digestion of M97-M99 and M101 (all pSB1A2+''cenA'') and M105 and M108 (pSB1A2+''cex'') with a) EcoRI, b) EcoRI/PstI, and M92 (pSB1A2+''cex'') with a) PstI, b) EcoRI/PstI. (06.08.08: Yan)
-
*Gel 44:* Double digests of BABEL1-glgc mut 1+2+3 (M117 to M120),&nbsp;pSB1A2-glgc mut 1+2 (M113-M116) and pSB1A2-CrtBI with EcoRI and PstI.(11.08.08: YAN)
+
'''Gel 39:''' Analytical digests using a) EcoRI, b) EcoRI/PstI of M70-M71 (pSB1A2+''glgC''-mut1,2) and M92 (pSB1A2+''cex''). Results do not look good. Bands are wrong size to correspond to vector and no bands match an expected insert size. (06.08.08: CF)<br />
-
*Gel 45:* P57 (cex from cells) EcoRI/PstI double digest, undigested P64 (cex from genomic DNA), P66 (zm1/ISA1) and P67 (zm2/ISA2). (12.08.08: CF/AM)
+
'''Gel 40''' PCR products of PcstA (P54) (and SOB). Results look promising (fairly prominent band <500bp). (07.08.08: CF)<br />
-
*Gel 46:* Double digests of M121 to M126: M121 to M123 (pSB1A2-CrtY-1 to pSB1A2-CrtY-3), M124 to M126 (pSB1A2-CrtY-RBS-1 to pSB1A2-CrtY-RBS-3) with EcoRI and PstI. (12.08.08: YAN)
+
'''Gel 41:''' PCR products of P55-P58 (''cenA'' and ''cex''): ''cex'' with KOD polymerase (P57) generated a single band of the desired size. However, ''cex'' with Velocity polymerase and ''cenA'' with both KOD or Velocity polymerase failed. (11.08.08: Yan)<br />
-
*Gel 47:* P57 and P64 (cex) PstI digests and P65 (cenA) (12.08.08: AM)
+
'''Gel 42:''' PCR products P61-P63 (''rrnB'', ''cenA'', ''cex''): Very clear band for ''rrnB'', no bands for either ''cenA'' or ''cex''. (11.08.08: CF)<br />
-
*Gel 48:* M109 and M110 (_crtBI_) BamHI/EcoRV digest, M115 and M116 (pSB1A2\+_glgC_\-mut1,2) HindIII digest M115 and M116 (pSB1A2\+_glgC_\-mut1,2) EcoRI/HindIII digest. (13.08.08: AH)
+
'''Gel 43:''' Double digests of BABEL2+''crtE'' (M79) and Edinbrick1 with EcoRI/PstI, showing a single band at ~3kb for BABEL2+''crtE'' digestion and bands at ~2.5kb and 800bp for Edinbrick1 digestion. (11.08.08: AH)<br />
-
*Gel 49:* P68 (cenB) and P69 (cenC) from _C. fimi_ genomic DNA. Failed. (13.08.08: AM)
+
'''Gel 44:''' Double digests of M109-M112 (pSB1A2+''crtB''+''crtI''), M113-M116 (pSB1A2+''glgC''-mut1,2) and M117-M120 (pSB1A2+''glgC''-mut1,2,3). M110-M112 (pSB1A2+''crtB''+''crtI'') have a band at 2.5kb as expected. M109 (pSB1A2+''crtB''+''crtI'') gave a band at 3kb instead, so should be ignored from now on.(11.08.08: Yan)<br />
-
*Gel 50:* M127~M138? Please confirm. (13.08.2008: OG, I believe, OG: i think this is the one when me andy and yan were loading it and taking pictures for the webpage.)
+
'''Gel 45:''' P57 (cex from cells) EcoRI/PstI double digest, undigested P64 (cex from genomic DNA), P66 (zm1/ISA1) and P67 (zm2/ISA2). (12.08.08: CF/AM)<br />
-
*Gel 51:* Undocumented gel stained with SYBR-safe with bands for purification?
+
'''Gel 46:''' Double digests of M121 to M126: M121 to M123 (pSB1A2-CrtY-1 to pSB1A2-CrtY-3), M124 to M126 (pSB1A2-CrtY-RBS-1 to pSB1A2-CrtY-RBS-3) with EcoRI and PstI. (12.08.08: YAN)<br />
-
*Gel 52:* P70 (glgC mut1+2), P71 (glgC mut 1+2+3). (14.08.2008: AM)
+
'''Gel 47:''' P57 and P64 (cex) PstI digests and P65 (cenA) (12.08.08: AM)<br />
-
*Gel 53:* Minipreps of M139 to M143 (CrtE+PSB1A2) (15.08.08: YAN/OG)
+
'''Gel 48:''' M109 and M110 (_crtBI_) BamHI/EcoRV digest, M115 and M116 (pSB1A2\+_glgC_\-mut1,2) HindIII digest M115 and M116 (pSB1A2\+_glgC_\-mut1,2) EcoRI/HindIII digest. (13.08.08: AH)<br />
-
*Gel 54:* P71A (glgC mut 1+2), P72 (glgC mut 1+2+3), P73~P77 (rrnB from _C. fimi_ genomic DNA) (15.08.08: AM)
+
'''Gel 49:''' P68 (cenB) and P69 (cenC) from _C. fimi_ genomic DNA. Failed. (13.08.08: AM)<br />
-
*Gel 55:* M144-M147 (pSB1A2+cenA), M148-M151 (pSB1A2+cex) and M152-M155 (pSB1A2+dxs+crtE) digested with EcoRI/PstI. (18.08.08: AH)
+
'''Gel 50:''' M127~M138? Please confirm. (13.08.2008: OG, I believe, OG: i think this is the one when me andy and yan were loading it and taking pictures for the webpage.)<br />
 +
'''Gel 51:''' Undocumented gel stained with SYBR-safe with bands for purification?<br />
 +
'''Gel 52:''' P70 (glgC mut1+2), P71 (glgC mut 1+2+3). (14.08.2008: AM)<br />
 +
'''Gel 53:''' Minipreps of M139 to M143 (CrtE+PSB1A2) (15.08.08: YAN/OG)<br />
 +
'''Gel 54:''' P71A (glgC mut 1+2), P72 (glgC mut 1+2+3), P73~P77 (rrnB from _C. fimi_ genomic DNA) (15.08.08: AM)<br />
 +
'''Gel 55:''' M144-M147 (pSB1A2+cenA), M148-M151 (pSB1A2+cex) and M152-M155 (pSB1A2+dxs+crtE) digested with EcoRI/PstI. (18.08.08: AH)<br />
 +
 
 +
Note: Remaining gels will be added when the relevant information is better organised.

Latest revision as of 21:30, 6 October 2008

< Back to Notebook

Gels

Gel 1: PCR reactions P1 (dxs), P2 (appY), P3 (glgC). (25.06.08)
Gel 2: EcoRI/PstI digests of M1 to M6 (pSB1A2+dxs). (30.06.08)
Gel 3: minipreps M2, M3, M4, M6, EcoRI, plus L1 and L2. (01.07.08)
Gel 4: minipreps M7 (BABEL1+glgC) and M8 to M12 (BABEL2+glgC), undigested. (01.07.08)
Gel 5: minipreps M7 to M12, EcoRI/PstI. (01.07.08)
Gel 6: minipreps M2, M3, M4, M6, HindIII; also P4 (BABEL1+glgC fusion PCR)and P5 (BABEL2+glgC fusion PCR). (01.07.08)
Gel 7: minipreps M10 and M11, EcoRI (two versions of this gel). (04.07.08)
Gel 8: PCR products P6 and P7 (mutagenic PCR on glgC [M11]). (04.07.08)
Gel 9: digests of maxipreps X1 (J33201) and X2 (pSB1A2+dxs). (08.07.08)
Gel 10: minipreps M13 to M18, EcoRI/PstI digests. (09.07.08)
Gel 11: M19 to M24 (BABEL2+glgC-mut2) EcoRI digests, plus PCR P11 (rbs+dxs). (10.07.08)
Gel 12: SacI/SpeI digests of M25 to M30 (possible rbs+dxs clones): none look right. Also shown is P12 (rbs+dxs fusion PCR product). (13.07.08)
Gel 13: PCR P13 and P14 to mutate out first EcoRI site from BABEL2+glgC-mut2 clones M19 and M22. (14.07.08)
Gel 14: minipreps M31 to M42 (pSB1A2+crtB and pSB1A2+crtI clones) EcoRI/PstI digests, and P15 (rbs+appY) (15.07.08)
Gel 15: redigest of M32, M36 (pSB1A2+crtB), M39, M40 (pSB1A2+crtI) with EcoRI/PstI; unsuccessful. (17.07.08)
Gel 16: purified PCR products for cenA, cenB, cenC, cex (P16 to P19); unsuccessful. (18.07.08)
Gel 17: PCR products for cenA, cenB, cenC, cex (P20 to P23); unsuccessful. (18.07.08)
Gel 18: EcoRI/PstI digests of M43 to M47 (BABEL2+glgC-mut1,2) and M48 (pSB1A2+rbs+dxs). M43 to M47 all look about right. M48 is ambiguous, could be incomplete deigestion, need to repeat. (19.07.08: CF)
Gel 19: P24 (cenA), P25 (cex) as well as SacI/SpeI digest of M48 (pSB1A2+rbs+dxs) and single (EcoRI) and double (EcoRI/PstI) digests of M36 (pSB1A2+crtB) and M42 (pSB1A2+crtI). (20.07.08: CF)
Gel 20: L15 (rbs+dxs with Edinbrick1) and L16 (rbs+appY with Edinbrick1) both failed. (22.07.08: OG, Yan)
Gel 21: P26 (rrnB from C. fimi); unsuccessful. (22.07.08: AM)
Gel 22: P27 (crtY, unsuccessful), P28 (pSB1A2+rbs+crtB, successful) and P29 (pSB1A2+rbs+crtI, successful). (23.07.08: AM, Yan, OG)
Gel 23: P30 (repeat rrnB from C. fimi) and pZntA, both successful. (23.07.08: AM)
Gel 24: Shows failure of P32-P35 (cenA, cenB, cenC and cex). (24.07.08: AM)
Gel 25: P36 (glgC-mut1,2 excision from BABEL2, successful), P37 (crtY, failed). (25.07.08: AH)
Gel 26: P38-41 (cenA, cenB, cenC, cex from heat-killed cell suspension) and P42-43 (cenA and cenB from 'impure' DNA solution). (28.07.08: AM)
Gel 27: Repeat of Gel 26 but stained with SYBRsafe. Putative cenA and cex fragments cut out. (29.07.08: AM)
Gel 28: M49-54 (pSB1A2+rbs+crtI) digested with EcoRI. (29.07.08: CF)
Gel 29: M55-60 (pSB1A2+pZntA), M61-66 (pSB1A2+rbs+CrtE) digested with EcoRI/PstI. Shows that ligations L23 and L24 (respectively) failed. (29.07.08: Yan, AM)
Gel 30: P46 (cenA), P47 (cex) from heat killed cell solution, annealing 65C. Gel shows failure of the PCR. (30/07/08)
Gel 31: P48 (crtY) and digests of M49 and M50 (pSB1A2-rbs+crtI) with a) XbaI, b) SpeI/XbaI, c) sac/speI, M63 (BABEL2+rbs+crtE) with a) EcoRI, b) EcoRI/PstI, M67 (pSB1A2+rbs+crtB) with a) EcoRI/PstI, b) sacI/speI. (30/07/08: OG)
Gel 32: M55-M60 (pSB1A2+pZntA) double digestion with EcoRI/PstI, and single digestion with EcoRI. (30.07.08: Yan)
Gel 33: M68-71 (pSB1A2+glgC-mut1,2) digested with EcoRI/PstI, M72-75 (pSB1A2+rbs+dxs) digested with EcoRI, M76-79 (BABEL2+crtE) digested with EcoRI/PstI. (01.08.2008: AM)
Gel 34: M68-69 (pSB1A2+glgC-mut1,2) digested with EcoRI alone and EcoRI+PstI, M72 (pSB1A2+rbs+dxs) digested with EcoRI alone and SacI/SpeI. (01.08.2008: AM)
Gel 35: P36 (glgC-mut1,2) and L25 (pSB1A2+glgC-mut1,2). P36 successful, L25 yields clear glgC-length band but faint vector-length band. (01.08.2008: AM)
Gel 36: P50 (glgC-mut1,2,3) and double digestion of M80-M96 with EcoRI/PstI. Results were good for M80-M82 (pSB1A2+rbs+appY), M84-M85 (pSB1A2+rbs+crtB), M92 (pSB1A2+cex) and P50. Other results were poor. (04.08.08: Yan, OG).
Gel 37: Double digestion of M97-M104 (pSB1A2+cenA) and M105-M108 (pSB1A2+cex) with EcoRI/PstI. Results were very poor apart from M97. (05.08.08: Yan)
Gel 38: Re-digestion of M97-M99 and M101 (all pSB1A2+cenA) and M105 and M108 (pSB1A2+cex) with a) EcoRI, b) EcoRI/PstI, and M92 (pSB1A2+cex) with a) PstI, b) EcoRI/PstI. (06.08.08: Yan) Gel 39: Analytical digests using a) EcoRI, b) EcoRI/PstI of M70-M71 (pSB1A2+glgC-mut1,2) and M92 (pSB1A2+cex). Results do not look good. Bands are wrong size to correspond to vector and no bands match an expected insert size. (06.08.08: CF)
Gel 40 PCR products of PcstA (P54) (and SOB). Results look promising (fairly prominent band <500bp). (07.08.08: CF)
Gel 41: PCR products of P55-P58 (cenA and cex): cex with KOD polymerase (P57) generated a single band of the desired size. However, cex with Velocity polymerase and cenA with both KOD or Velocity polymerase failed. (11.08.08: Yan)
Gel 42: PCR products P61-P63 (rrnB, cenA, cex): Very clear band for rrnB, no bands for either cenA or cex. (11.08.08: CF)
Gel 43: Double digests of BABEL2+crtE (M79) and Edinbrick1 with EcoRI/PstI, showing a single band at ~3kb for BABEL2+crtE digestion and bands at ~2.5kb and 800bp for Edinbrick1 digestion. (11.08.08: AH)
Gel 44: Double digests of M109-M112 (pSB1A2+crtB+crtI), M113-M116 (pSB1A2+glgC-mut1,2) and M117-M120 (pSB1A2+glgC-mut1,2,3). M110-M112 (pSB1A2+crtB+crtI) have a band at 2.5kb as expected. M109 (pSB1A2+crtB+crtI) gave a band at 3kb instead, so should be ignored from now on.(11.08.08: Yan)
Gel 45: P57 (cex from cells) EcoRI/PstI double digest, undigested P64 (cex from genomic DNA), P66 (zm1/ISA1) and P67 (zm2/ISA2). (12.08.08: CF/AM)
Gel 46: Double digests of M121 to M126: M121 to M123 (pSB1A2-CrtY-1 to pSB1A2-CrtY-3), M124 to M126 (pSB1A2-CrtY-RBS-1 to pSB1A2-CrtY-RBS-3) with EcoRI and PstI. (12.08.08: YAN)
Gel 47: P57 and P64 (cex) PstI digests and P65 (cenA) (12.08.08: AM)
Gel 48: M109 and M110 (_crtBI_) BamHI/EcoRV digest, M115 and M116 (pSB1A2\+_glgC_\-mut1,2) HindIII digest M115 and M116 (pSB1A2\+_glgC_\-mut1,2) EcoRI/HindIII digest. (13.08.08: AH)
Gel 49: P68 (cenB) and P69 (cenC) from _C. fimi_ genomic DNA. Failed. (13.08.08: AM)
Gel 50: M127~M138? Please confirm. (13.08.2008: OG, I believe, OG: i think this is the one when me andy and yan were loading it and taking pictures for the webpage.)
Gel 51: Undocumented gel stained with SYBR-safe with bands for purification?
Gel 52: P70 (glgC mut1+2), P71 (glgC mut 1+2+3). (14.08.2008: AM)
Gel 53: Minipreps of M139 to M143 (CrtE+PSB1A2) (15.08.08: YAN/OG)
Gel 54: P71A (glgC mut 1+2), P72 (glgC mut 1+2+3), P73~P77 (rrnB from _C. fimi_ genomic DNA) (15.08.08: AM)
Gel 55: M144-M147 (pSB1A2+cenA), M148-M151 (pSB1A2+cex) and M152-M155 (pSB1A2+dxs+crtE) digested with EcoRI/PstI. (18.08.08: AH)

Note: Remaining gels will be added when the relevant information is better organised.

Retrieved from "http://2008.igem.org/Edinburgh/Notebook/Gels"