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| - | <html lang="en">
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| - | <head>
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| - | <title>Notebook</title>
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| - | <script src="https://googledrive.com/host/0B4ZBZOYYKBzETkFqdnhMeV9fMzA" ></script>
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| - | <script src="https://googledrive.com/host/0B4ZBZOYYKBzEZTdBSFdUV19LYjQ" type="text/javascript"></script>
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| - | <script src="https://googledrive.com/host/0ByCIrEV11SKjaFJSb0FVU1pmdk0/" type="text/javascript"> </script> <!--debounce-->
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| - | <script>
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| - | $(document).ready(function($) {
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| | | | |
| - | /*load in the sidebar*/
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| - | $('.left_wrap').load('https://googledrive.com/host/0B4ZBZOYYKBzEclFHMmpZcVlydmc');
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| - |
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| - | });
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| - |
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| - | /*
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| - | *
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| - | *javascript for Penn's iGEM wiki
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| - | *
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| - | */
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| - |
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| - | /*script for all pages*/
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| - | jQuery(document).ready(function($) {
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| - |
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| - | //when the mouse hovers over project or about, show the corresponding menu
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| - | $(".dropdown").mouseenter(function(){
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| - | $(this).find(".dropdown-menu").show();
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| - | });
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| - |
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| - | //when the mouse clicks a notebook tab, the notebook flips to that tab
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| - | var $items = $('#vtab>ul>li');
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| - | $items.click(function() {
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| - | $items.removeClass('selected');
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| - | $(this).addClass('selected');
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| - |
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| - | var index = $items.index($(this));
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| - | $('#vtab>div').hide().eq(index).show();
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| - | }).eq(0).click();
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| - |
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| - |
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| - | //when the mouse leaves project or about, hide the corresponding drop down menu
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| - | $(".dropdown, #drop-menu1,#drop-menu2").mouseleave(function(){
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| - | $(this).find(".dropdown-menu").hide();
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| - | });
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| - |
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| - | //when mouse enters a button on the sidebar, change it to active
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| - | //(changes the background color)
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| - | $(".dropdown li").mouseenter(function(){
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| - | $(this).addClass("active");
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| - | });
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| - |
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| - | //When the mouse leaves the button, remove the active class
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| - | $(".dropdown li").mouseleave(function(){
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| - | $(this).removeClass("active");
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| - |
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| - | });
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| - |
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| - | /*
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| - | $(".left_wrap").mouseleave(function(){
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| - | $(".drop-menu-wrap").css("display", "none !important");
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| - | $(".navbar").hide();
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| - | $(".left_wrap").animate({
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| - | width: "65px"
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| - | }, 250, function() {
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| - | });
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| - |
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| - | });
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| - |
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| - | //when the mouse enters the sidebar, show it and increase the width
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| - | $(".left_wrap").mouseenter(function(){
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| - | $(".drop-menu-wrap, #drop-menu1, #drop-menu2").css("display", "inline !important");
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| - | $(".left_wrap").animate({
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| - | width: "200px"
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| - | }, 250, function() {
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| - | $(".navbar").show();
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| - |
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| - | });
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| - | });
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| - | */
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| - |
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| - |
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| - | });
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| - | </script>
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| - |
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| - | <style>
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| - | /************
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| - | *Stylesheet for Penn's iGEM wiki
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| - | *
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| - | **********/
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| - |
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| - | /*import the font quicksandlight (using this for > arrow)*/
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| - | @font-face
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| - | {
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| - | font-family: QuickSandLight;
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| - | src: url('https://googledrive.com/host/0B4ZBZOYYKBzEazRybm9PV21QdTA'),
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| - | }
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| - |
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| - | /*format paragraph, h1, h2, and h3*/
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| - |
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| - | p {
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| - | color: black; /*font color*/
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| - | font-family: arial, sans-serif; /*font is arial, with sans-serif as a back up*/
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| - | font-size: 13px;
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| - | }
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| - |
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| - | h1, h2 {
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| - | color: black; /*font color*/
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| - | font-family: arial, sans-serif;/*font is arial, with sans-serif as a back up*/
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| - | font-size: 15px;
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| - | }
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| - |
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| - | /*h3 will be the same as h1 and h2, but it will be centered*/
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| - | h3 {
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| - | color: black;
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| - | font-family: arial, sans-serif; /*font is arial, with sans-serif as a back up*/
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| - | font-size: 15px;
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| - | text-align: center; /*center*/
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| - |
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| - | }
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| - |
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| - | /*links in sidebar*/
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| - | a{
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| - | font-family: arial, sans-serif;
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| - | text-decoration: none; /*gets rid of underlines*/
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| - | color: black;
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| - | }
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| - |
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| - | /*visited link same as regular link*/
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| - | a:visited {
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| - | color: black !important;
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| - | text-decoration: none; /*gets rid of underlines*/
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| - | }
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| - |
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| - | /*link when the mouse hovers over it*/
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| - | a:hover {
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| - | color: #07304b !important; /*font color is dark blue*/
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| - | text-decoration: none; /*gets rid of underlines*/
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| - | }
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| - |
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| - | .navbar ul {
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| - | list-style-type: none; /*gets rid of bullets*/
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| - | }
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| - |
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| - | /*sidebar--fix line heights to get some padding. change fonts */
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| - | .navbar{
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| - | position: relative;
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| - | list-style-type: none; /*no bullets*/
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| - | line-height: 30px; /*changes line height to give the sidebar links some padding*/
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| - | font-size: 15px;
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| - | font-family: arial, sans-serif;
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| - | }
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| - |
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| - | .navbar li {
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| - | /*not actually sure if this is neccessary, but I'll just leave it here*/
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| - | padding-left: 50px;
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| - | margin-left: -50px;
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| - | }
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| - |
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| - | /*sidebar--change the color of the background of the link on the sidebar*/
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| - | .navbar li:hover {
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| - | width: 100%; /*expand the link to the width of the sidebar.*/
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| - | background-color: rgb(200, 200, 215); /*change background color to light blue*/
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| - | }
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| - |
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| - | /*change color of the link that's active*/
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| - | .active {
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| - | color: #07304b !important;
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| - | }
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| - |
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| - | /*get rid of default list formatting*/
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| - | .dropdown{
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| - | list-style-type: none; /*no bullets or numbering*/
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| - |
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| - | }
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| - | .dropdown-menu{
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| - | list-style-type: none; /*no bullets or numbering*/
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| - | }
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| - | /*change the background color of the active link*/
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| - | .dropdown-menu .active {
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| - | background-color: rgb(200,200,215); /*light blue background color*/
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| - | width: 100px !important; /*set the width*/
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| - | padding: 5px;
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| - | margin: 0px !important; /*get rid of margins*/
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| - | }
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| - |
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| - | /*format the arrow that is on the project and about links*/
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| - | .arrow{
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| - | display: inline; /*inline so it stays next to the link*/
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| - | float: right; /*move it to the right*/
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| - | margin-right: 20px; /*but not all the way to the right*/
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| - | font-size: 20px; /*make it bigger*/
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| - | font-family: QuickSandLight, arial, sans-serif; /*quicksand light seems to make the arrows wider looking*/
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| - |
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| - | }
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| - |
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| - | /*format each button on the sidebar drop down menus*/
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| - | .dropdown-menu li {
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| - | background-color: white; /*white background*/
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| - | width: 100px; /*set width*/
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| - | margin: 0px; /*no margins*/
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| - | padding: 5px;
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| - | }
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| - |
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| - | /*position the dropdown menus so they line up with the corresponding link. It's a little tricky because the positioning is absolute--it's a
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| - | matter of eyeballing. This is probably not the most efficient way of doing this, but since there's only two dropdowns it works*/
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| - |
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| - | /*drop-menu1 is for the project dropdown*/
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| - | #drop-menu1{
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| - | position: absolute; /*absolutely position so it doesn't conflict with other content*/
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| - | left: 155px; /*move it left until it's not in the navbar anymore*/
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| - | top: 27px; /*move it down right next to the project button (higher number is lower on page)*/
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| - | text-align: center; /*center the text*/
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| - | display: inline;
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| - | line-height: 20px; /*give it a big line height so it has some padding*/
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| - | z-index: 9999; /*bring it up front so it's not behind anything*/
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| - | display: none; /*hide it at first*/
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| - | }
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| - |
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| - | /*drop-menu2 is for about (comments are same as above)*/
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| - | #drop-menu2{
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| - | width: 100px;
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| - | position: absolute;
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| - | left: 155px;
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| - | top: 59px; /*move it down next to the about button (higher number is lower on page)*/
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| - | text-align: center;
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| - | display: inline;
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| - | line-height: 20px;
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| - | display: none;
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| - | z-index: 9999;
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| - |
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| - | }
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| - |
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| - | /*wrap the drop-menu to make it a box--this wrap actually is not very important
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| - | I actually am not sure if it still does anything, but I'm leaving it just in case it does*/
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| - | .drop-menu-wrap{
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| - | width: 120px; /*give it a width*/
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| - | background-color: white;
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| - | text-align: center; /*center the text*/
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| - | min-height: 700px; /*give it a minimum height*/
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| - | float: left; /*float it to the left*/
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| - | margin-left: 0px;
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| - | }
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| - |
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| - |
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| - | /*format the left wrapper (sidebar).
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| - | fixed left is not being used, but I'm keeping it there just in case*/
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| - | .left_wrap, .fixed-left {
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| - | margin-left: -15px; /*this makes sure there's no margin between the sidebar and the page*/
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| - | padding-top: 80px; /*padding to the top so the links don't start there*/
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| - | width: 200px; /*give it a width*/
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| - | min-height: 100%; /*height should take up entire height*/
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| - | position: fixed; /*fix it so it stays in place when you scroll*/
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| - | background-color: white;
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| - | /*cross browser opacity*/
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| - | -ms-filter: "progid:DXImageTransform.Microsoft.Alpha(Opacity=80)";
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| - | filter: alpha(opacity=80);
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| - | -moz-opacity: 0.8;
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| - | -khtml-opacity: 0.8;
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| - | opacity: 0.8;
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| - | z-index: 999; /*make sure sidebar is over everything else in case there is some overlap*/
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| - |
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| - | }
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| - |
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| - |
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| - | /*position the igem logo, resize, and fix it*/
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| - | #igem {
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| - | z-index: 999; /*it should be over everything in case there is overlap*/
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| - | height: 50px; /*fix the height and leave the width at auto*/
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| - | top: 45px; /*position it 45 pixels down*/
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| - | right: 35px; /*and 35 to the right*/
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| - | margin: 30px 15px 15px 15px;
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| - | position: fixed; /*fix it in place so it doesn't move when you scroll*/
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| - | /*give it some transparency (cross browser)*/
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| - | -ms-filter: "progid:DXImageTransform.Microsoft.Alpha(Opacity=80)";
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| - | /* IE 5-7 */
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| - | filter: alpha(opacity=80);
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| - | /* Netscape */
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| - | -moz-opacity: 0.8;
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| - | /* Safari 1.x */
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| - | -khtml-opacity: 0.8;
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| - | /* Good browsers */
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| - | opacity: 0.8;
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| - |
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| - | }
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| - |
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| - | /*position the penn logo, resize, and fix it (same comments as above)*/
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| - | #penn {
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| - | z-index: 999;
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| - | height: 40px;
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| - | top: -5px;
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| - | right: 20px;
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| - | margin: 30px 15px 15px 15px;
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| - | position: fixed;
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| - | -ms-filter: "progid:DXImageTransform.Microsoft.Alpha(Opacity=80)";
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| - | /* IE 5-7 */
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| - | filter: alpha(opacity=80);
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| - | /* Netscape */
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| - | -moz-opacity: 0.8;
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| - | /* Safari 1.x */
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| - | -khtml-opacity: 0.8;
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| - | /* Good browsers */
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| - | opacity: 0.8;
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| - | }
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| - |
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| - |
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| - | /*******Format background image, padding, margins of background
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| - | ******************/
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| - | .section1 {
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| - | position: relative;
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| - | bottom: 0px;
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| - | padding: 0;
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| - | margin: 0;
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| - | height: 100%;
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| - | z-index: 998;
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| - | margin-left: -15px; /*make sure there's no space between the browser edge and background*/
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| - | padding-left: 200px; /*padding is as big as the sidebar so there's no overlap and it centers without taking that into account*/
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| - | padding-top: 75px; /*give it some padding*/
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| - | overflow: hidden;
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| - | }
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| - |
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| - |
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| - | /*****
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| - | putting the background image in the body instead of section1 to fix gray bar issue
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| - | ******/
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| - | html, body {
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| - | background: url('https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/EQUBpSyRCy/jake%20photo/DSC_0096.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg') no-repeat center center fixed; /* and add background image center it*/
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| - | /* min-height: 100%; /*make sure it takes up full screen*/
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| - | /*the next few are for cross browser background size. "cover" makes the image cover the entire container*/
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| - | -webkit-background-size: cover;
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| - | -moz-background-size: cover;
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| - | -o-background-size: cover;
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| - | background-size: cover;
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| - | }
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| - |
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| - |
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| - | /*sets margins, padding and width just in case*/
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| - | html,body{
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| - | width: 100%;
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| - | margin: 0;
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| - | padding: 0;
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| - |
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| - | }
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| - | /*white text box - margins are auto.
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| - | *Because of padding in the section divs, it should align taking the navbar into account.
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| - | *Use this around content
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| - | */
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| - | .text {
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| - | color: black;
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| - | width: 700px;
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| - | /*the textbox should extend if text goes over this height, but I noticed it doesn't always.
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| - | Hard code the min-height into the specific textbox's html to fix this issue if it occurs*/
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| - | min-height: 500px;
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| - | background: rgb(54, 25, 25); /* Fall-back for browsers that don't
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| - | support rgba */
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| - | background: rgba(255, 255, 255, .8);
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| - | margin: auto;
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| - | overflow: hidden;
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| - | padding: 20px;
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| - | margin-top: 20px;
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| - | -moz-border-radius: 10px;
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| - | -webkit-border-radius: 10px;
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| - | -khtml-border-radius: 10px;
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| - | border-radius: 10px;
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| - | }
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| - |
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| - |
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| - | /*get rid of gray bars on top and bottom. This accounts for some of the leftover wiki formatting*/
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| - | #content {
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| - | margin-top: -10px;
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| - | margin-bottom: -20px;
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| - | padding: 0px;
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| - | }
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| - |
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| - |
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| - | /*wrap any number of text columns that add up to less than 750px wide (plus margins), float one left, one right,
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| - | *one or more in the middle (adjust the middle ones' margins until centered or float center if there's just one.
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| - | *The text wrap will keep the margins on either side centered.
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| - | */
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| - | .text-wrap {
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| - |
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| - | overflow: hidden;
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| - | width: 750px;
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| - | margin: auto;
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| - | }
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| - |
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| - | /*give the textboxes for methods a different minimum height and margin*/
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| - | #methods {
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| - | min-height: 50px !important; /*change this if this is to tall/short*/
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| - | margin-bottom: 10px;
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| - | }
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| - |
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| - |
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| - | /*format the title of the sections in the sidebar and turn them 90 degrees*/
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| - | .section-title {
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| - | top: 50% !important;
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| - | left: 2px;
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| - | position: fixed !important;
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| - | font-family: arial, sans-serif;
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| - | font-size: 25px;
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| - | -webkit-transform: rotate(-90deg);
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| - | -moz-transform: rotate(-90deg);
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| - | -o-transform: rotate(-90deg);
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| - | -ms-transform: rotate(-90deg);
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| - | transform: rotate(-90deg);
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| - | z-index: 9999; /*keep it above everything else*/
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| - | }
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| - |
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| - |
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| - |
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| - |
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| - | /****************
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| - | Getting rid of wiki defaults
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| - | *****************/
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| - |
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| - | .outer {
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| - | width: 100%;
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| - | text-align: center;
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| - | float: center;
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| - | height: 100%;
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| - | background-color: white;
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| - | }
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| - |
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| - | #p-logo {
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| - | position: absolute;
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| - | display: none;
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| - | }
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| - |
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| - | #content {
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| - | width: 100%;
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| - | background-color: white;
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| - | }
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| - |
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| - | #top-section {
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| - | position: absolute;
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| - | }
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| - |
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| - | .firstHeading {
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| - | position: absolute;
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| - | display: none;
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| - | }
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| - |
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| - | /*.thumb {
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| - | display: none;
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| - | position: absolute;
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| - | }*/
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| - |
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| - |
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| - | #search-controls {
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| - | display: none;
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| - | position: absolute;
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| - | }
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| - |
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| - |
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| - | #footer {
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| - | display: none;
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| - | position: absolute;
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| - | }
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| - |
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| - | ul {
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| - | list-style-type: none;
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| - | list-style-image: none;
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| - | }
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| - |
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| - | .printfooter {
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| - | display: none;
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| - | position: absolute;
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| - | }
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| - |
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| - |
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| - | .visualClear {
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| - | display: none;
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| - | position: absolute;
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| - | }
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| - |
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| - | #footer-box {
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| - | display: none;
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| - | position: absolute;
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| - | }
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| - |
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| - |
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| - | body {
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| - | margin: 10px 0 0 0;
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| - | padding: 0;}
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| - | #top-section {
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| - | width: 965px;
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| - | height: 0;
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| - | margin: 0 auto;
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| - | padding: 0;
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| - | border: none;}
| |
| - | body {
| |
| - | margin: 10px 0 0 0;
| |
| - | padding: 0;}
| |
| - | #top-section {
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| - | width: 965px;
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| - | height: 0;
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| - | margin: 0 auto;
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| - | padding: 0;
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| - | border: none;}
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| - | #menubar {
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| - | font-family: arial, sans-serif;
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| - | z-index: 9999;
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| - | font-size: 65%;
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| - | top: 5px;
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| - | height: 50px;}
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| - | .left-menu:hover {
| |
| - | background-color: transparent;}
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| - | #menubar li a {
| |
| - | background-color: transparent;}
| |
| - | #menubar:hover {
| |
| - | color: white;}
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| - | #menubar li a {
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| - | #menubar {
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| - | list-style-type: none;
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| - | </style>
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| - | </head>
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| - | <body>
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| - | <img src="http://upload.wikimedia.org/wikipedia/en/d/d6/IGEM_official_logo.png" id="igem"/><!--igem logo-->
| |
| - | <img src="http://collegediabetesnetwork.org/wp-content/uploads/2012/07/UPenn_logo1.png" id="penn"/> <!--penn logo-->
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| - | <div class="left_wrap">
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| - |
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| - |
| |
| - | <!-- <div class="logo-wrap"><img src="https://googledrive.com/host/0B4ZBZOYYKBzEUlI3ZDU2OGRrc1E" id="penn"/></div><!--penn logo-->
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| - |
| |
| - |
| |
| - | <a href="https://2013.igem.org/Team:Penn/HumanPractices"><li>human practices</li></a>
| |
| - | <a href="https://2013.igem.org/Team:Penn/Notebook"><li class="active" style="background-color: rgb(200,200,215);">notebook</li></a>
| |
| - | </ul>
| |
| - |
| |
| - |
| |
| - | <div class="section-title">notebook</div>
| |
| - |
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| - | </div>
| |
| - | <div class="section1" style="background-position: top;">
| |
| - | <div class="text">
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| - |
| |
| - | <div id="vtab">
| |
| - | <ul id="tab_list">
| |
| - | <li>Week 1</li>
| |
| - | <li>Week 2</li>
| |
| - | <li>Week 3</li>
| |
| - | <li>Week 4</li>
| |
| - | <li>Week 5</li>
| |
| - | <li>Week 6</li>
| |
| - | <li>Week 7</li>
| |
| - | <li>Week 8</li>
| |
| - | <li>Week 9</li>
| |
| - | <li>Week 10</li>
| |
| - | <li>Week 11</li>
| |
| - | <li>Week 12</li>
| |
| - | <li>Week 13</li>
| |
| - | <li>Week 14</li>
| |
| - | <li>Week 15</li>
| |
| - | <li>Week 16</li>
| |
| - | <li>Week 17</li>
| |
| - | <li>Week 18</li>
| |
| - |
| |
| - | </ul>
| |
| - |
| |
| - |
| |
| - | <div>
| |
| - | <h2>WEEK 1</h2>
| |
| - | <div class="box">
| |
| - | <h2>June 4 2013 - June 11 2013</h2>
| |
| - | <img src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/APrj2Se2i-/Lynch%20Labs%20PGFI/BE_LynchLabGroup_DSC2811.jpg?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg" class="image"/><li>4-Jun
| |
| - | <ol>
| |
| - | <li>Learned how to make competent cells, growing up two strains for tomorrow</li>
| |
| - | <li>Transformed 8 plasmids</li>
| |
| - | <li>Determined EL222 fusion is risky but still going ahead with it</li>
| |
| - | <li>Linkers are totally setlled</li>
| |
| - | <li>Found zinc finger plasmid and updated target sequence</li>
| |
| - | <li>Learned how to make tetr- mcherry fusion</li>
| |
| - | <li>Settled on 5 promoters</li>
| |
| - |
| |
| - | </ol></li>
| |
| - |
| |
| - | <br/>
| |
| - | <li>5-Jun
| |
| - | <ol>
| |
| - | <li>Learned how to make competent cells, testing them and then making more tomorrow</li>
| |
| - | <li>Transformed 8 plasmids again</li>
| |
| - | <li>Made primers to clone the TET-GFP reporter system, the mCherry promoter strength system,
| |
| - | ready to order</li>
| |
| - | <li>Made ultramers for variable promoter blocks (and no target neg controls) – ready to order</li>
| |
| - | <li>Spilled a lot of iced tea outside, bummer</li>
| |
| - | <li>Started primers for dna binding machines</li>
| |
| - | <li>Got a handle on cas9 fusions (pun intended).</li>
| |
| - | <li>Put awesome pics in dropbox</li>
| |
| - |
| |
| - | </ol></li>
| |
| - | <br/>
| |
| - | <li>6-Jun
| |
| - | <ol>
| |
| - | <li>Clean up dropbox</li>
| |
| - | <li>Update budget sheet with addgene and cell center orders</li>
| |
| - | <li>Finish primers for fusion</li>
| |
| - | <li>Set up plate reader for GFP and mCherry assays</li>
| |
| - | <li>run minipreps on pdawn, pdawn-mcherry, pet26b</li>
| |
| - | <li>Grow up mCherry stock</li>
| |
| - | <li>Wrote Penn iGEM on our plasmid</li>
| |
| - | </ol></li>
| |
| - | <br/>
| |
| - | <li>7-Jun
| |
| - | <ol>
| |
| - | <li>Transform</li>
| |
| - | <ol>
| |
| - | <li>C0012 –amp/chlor (do both)</li>
| |
| - | <li>M11307 – amp/chlor (do both)</li>
| |
| - | <li>I13458 – amp/chlor (do both)</li>
| |
| - | <li>R0010 – amp/chlor (do both)</li>
| |
| - | <li>R0051 – amp</li>
| |
| - | <li>K206000 –chlor</li>
| |
| - | </ol>
| |
| - | <li>Start the LIMS and file all the strains and DNA we have made/ ordered</li>
| |
| - |
| |
| - | </ol></li>
| |
| - | <br/>
| |
| - | <li>8-Jun</li> <ol>
| |
| - | <li>Miniprep Addgene stuff + transformations that worked</li>
| |
| - | <li>Growing up low copy plasmids in 40mLs</li>
| |
| - | </ol> <li>Mini-prepped</li><ol>
| |
| - | <li>I9002</li>
| |
| - | <li>I13458</li>
| |
| - | <li>C0051</li>
| |
| - | <li>Pdawn-mcherry</li>
| |
| - | <li>Pdawn</li>
| |
| - | <li>Dhsa mcherry</li>
| |
| - | <li>Pdawn dhsa</li>
| |
| - | <li>Psb1a3</li>
| |
| - | <li>JM mcherry</li>
| |
| - | <li>Pet26b</li>
| |
| - |
| |
| - | </ol> </div>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div>
| |
| - | <h2>WEEK 2</h2>
| |
| - |
| |
| - | <div class="box">
| |
| - | <h2>June 11 2013 - June 18 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/-ZtRJ7Gu1U/photo-8.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - | <li>17-Jun <ol>
| |
| - | <li>Grow up luxI culture and grow up tetR culture </li>
| |
| - | <li>Sequence all of the minipreps</li>
| |
| - | <li>Transform t9002 in psb1A3 in NEB10</li>
| |
| - | <li>Retransform ptetGFP to see if BL21DE3 cells are competent </li>
| |
| - | <li>Transform r0079, k081015, r0063 in NEB10 </li>
| |
| - | <li>Miniprep psb1k3</li>
| |
| - | <li>Redo dam gel with more dna </li>
| |
| - | <li>Figure out second control zfp from addgene </li>
| |
| - | <li>Figure out how to add luxR binding site to target region </li>
| |
| - | <li>Order sequencing primers for all addgene minipreps </li>
| |
| - | <li>Bisulfite converted msssi methylated c0051</li>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | </ol></li>
| |
| - |
| |
| - | <br/>
| |
| - | <li>18-Jun<ol>
| |
| - | <li>The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results</li>
| |
| - | <li> Miniprep c0078, c0079 + make glycerol stocks check same plasmid with our kit </li>
| |
| - | <li>Order 13420 (second zfp)</li>
| |
| - | <li>Design a way to make variable promoters more easily varied (for a biobrick so teams can use the reporter plasmid for their own nefarious reasons)</li>
| |
| - | </ol></li>
| |
| - |
| |
| - | </br>
| |
| - |
| |
| - | <li>19-Jun <ol>
| |
| - | <li>Transform failed transformation</li>
| |
| - | <li> Make competent DH5a && Dam- </li>
| |
| - | <li>Figure out methylation assays for promoters</li>
| |
| - | <li>Miniprep psb1A3 && all the 40mL cultures</li>
| |
| - | <li>Picked many colonies</li>
| |
| - | <li>Check pTet-gfp under blue light</li>
| |
| - |
| |
| - | </ol></li> </div></div>
| |
| - |
| |
| - |
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 3</h2>
| |
| - | <div class="box"> <h2>June 18 2013 - June 25 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>21-Jun<ol>
| |
| - | <li>troubleshoot plux-luxI pcr</li>
| |
| - | <li>roubleshoot pdawn-luxI pcr</li>
| |
| - | <li>made pDawn-tetR pcr work</li>
| |
| - | <li>troubleshoot pet26b-tetR pcr</li>
| |
| - | <li>troubleshoot pDawn-GFP pcr</li>
| |
| - | <li>troubleshoot pDawn-mCherry-secretion tag pcr </li>
| |
| - | <li>miniprep growing cultures, be sure to pick only the glowing ligations </li>
| |
| - | <li>ransform the correct t9002 amp ligation - determined from gel</li>
| |
| - | <li>digested t9002 in amp and ptet gfp in amp to identify the correct ligation</li>
| |
| - | <li>all of the chosen ptet gfp ligations worked, but let's not use 5 || 13 12. troubleshoot t9002 digest</li>
| |
| - | <li>troubleshoot t9002 digest</li><ol>
| |
| - | <li>check for contamination of something (run uncut sample, sample + buffer, sample + 1
| |
| - | enzyme, sample + other enzyme, sample + both enzymes)</li>
| |
| - | </ol>
| |
| - | </ol></li>
| |
| - |
| |
| - | <br/>
| |
| - | <li>24-Jun<ol>
| |
| - | <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>
| |
| - | <li>Digested/ligated/transformed t9002 in amp</li>
| |
| - | <li>Get methylated biobrick sequenced</li>
| |
| - | <li>get chlor backbones sequenced</li>
| |
| - | <li>culture t9002 transformations in liquid media with i751250</li>
| |
| - | <li>mini prep stuff in the incubator</li>
| |
| - | <li>figure out the primer issues 8</li>
| |
| - | <li> Pick t9002 colonies for miniprep </li>
| |
| - | </ol></li></div>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div>
| |
| - | <h2>WEEK 4</h2>
| |
| - | <div class="box"> <h2>June 25 2013 - July 2 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/1LlS0hsVIZ/Screen%20shot%202013-05-29%20at%2016.22.13.png?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - | <ul><li>26-Jun<ol>
| |
| - | <li>Dam-/dh5a v dam methylation + dpnI && dpnII digest</li>
| |
| - | <li>Digested/ligated/transformed t9002 in amp </li>
| |
| - | <li>get chlor backbones sequenced</li>
| |
| - | <li>culture t9002 transformations in liquid media with i751250 </li>
| |
| - | <li>mini prep stuff in the incubator</li>
| |
| - | <li>figure out the primer issues</li>
| |
| - | <li> Pick t9002 colonies for miniprep</li>
| |
| - | <li>USER Cloning reporter plasmid </li>
| |
| - | </ol></li>
| |
| - | <br/>
| |
| - |
| |
| - | <li>1-Jul<ol>
| |
| - | <li>Beautiful Brady Bunch photoshoot</li>
| |
| - | <li>Troubleshooted and Re-tred PCR for user ends for reporter plasmid</li>
| |
| - | <li>Called IDT about pcr assembly – they said Gibson tends to work better, no mutations, all in one tub. If we must PCR assembly – add DMSO, hotstart reaction, anneal at 68-70C (we did this).</li>
| |
| - | <li>Get methylated biobrick sequenced </li>
| |
| - | <li>Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11 </li>
| |
| - | <li> Check if plux/luxI system is working in liquid cultures – this failed</li> <ol style =""margin-left: 50px;"">
| |
| - | <li>a. Might be strain competition, need to know growth rates</li>
| |
| - | </ol>
| |
| - | <li>Re-suspend primers for lux amplifier </li>
| |
| - | <li>Mini-prep: e0040, psb1a3, r0062</li>
| |
| - | </ol></li>
| |
| - | <br/>
| |
| - |
| |
| - | <li>2-Jul <ol><br>
| |
| - | <li>Think about application of mathylation project in e.coli</li>
| |
| - | <li>Ceck if plux/GFP-psb1C3 system is working in liquid cultures
| |
| - | <ol>
| |
| - | <li>+/- AHL induction at 100nM</li>
| |
| - | <li>Compare with ptetGFP fluorescence, normal LB fluorescence</li>
| |
| - | </ol></li>
| |
| - | <li>Streak zinc finger 2</li>
| |
| - | <li>Grow up 44251</li>
| |
| - | <li>Transform up R0062</li>
| |
| - | <li>When BstuI arrives<ol>
| |
| - | <li>Assay BstuI working</li>
| |
| - | <li>Assay the bvluc and tale1 minipreps in dh5a, dam-, and bl21 cells with msssi and bstuI </li>
| |
| - | <li>Results: BstUI is blocked by methylation, and cells don’t normally methylate</li>
| |
| - | </ol></li>
| |
| - | <li>Growing up t9002 in chlor and i751250 in amp for fluorescence study</li>
| |
| - | <li>Investigate CHIP or other ways of determining DNA binding domain specificity </li>
| |
| - | </ol></li></ul></div><!--/box-->
| |
| - | </div>
| |
| - |
| |
| - | <div>
| |
| - | <h2>WEEK 5</h2>
| |
| - | <div class="box"> <h2>July 2 2013 - July 9 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Y5d0PLu26o/photo-7.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - | <ul><li>3-Jul<ol>
| |
| - | <li>Streak zinc finger 2 </li>
| |
| - | <li>Grow up 44251 </li>
| |
| - | <li>Look into lux box being light sensitive </li>
| |
| - | </ol></li>
| |
| - |
| |
| - | <li>4-Jul<ol>
| |
| - | <li>Mini prep 44251 </li>
| |
| - | <li>Pick ZFP2 colonies to grow up, throw out liquid culture in fridge </li>
| |
| - | </ol></li>
| |
| - |
| |
| - | <li>5-Jul<ol>
| |
| - | <li>Repeat BstUI assay, taking into account new controls</li>
| |
| - | <li>Suspend the primers in the freezer </li>
| |
| - | <li>We need to check if the origin of replications are compatible before co transformation</li>
| |
| - | <li>Characterize pDawn-mcherry </li>
| |
| - | <li>Practice measuring fluorescence</li>
| |
| - | </ol></li>
| |
| - |
| |
| - |
| |
| - | <li>6-Jul
| |
| - | <ol>
| |
| - | <li>troubleshoot ptetGFP user PCR - band was visible but too small to extract</li>
| |
| - | <li>gel extract promoter fragments from USER PCR</li>
| |
| - | <li>re-do USER PCR for: TetR, pTetGFP</li>
| |
| - | <li>Design/check/order (Tale,Cas,ZFP)-flex-REV primers - check fwd primers</li>
| |
| - | </ol></li>
| |
| - |
| |
| - |
| |
| - | <li>8-Jul<ol>
| |
| - | <li>pick up minigenes and primers from the cell center</li>
| |
| - | <li>pick many colonies, colony PCR, and run results on a gel</li>
| |
| - | <li>Restriction digest sgRNA with RFP for cotransformation with cas9 </li>
| |
| - | <li>Find/make/buy TBE for use in TBE gels (hi-resolution)</li>
| |
| - | <li>PCR assemble MsssI with USER primers</li>
| |
| - | <li>get pTetGfp from pcr box and run gel</li>
| |
| - | <li>PCR purify pTetGFP in eppendorf and LIMs it </li>
| |
| - | <li>Fab device(s) and begin fiddling with them</li>
| |
| - | <li>get t9002 seq</li>
| |
| - | <li>pcr luxI, gel verify, pcr purify, restriction digest, ligate into pDawn (remember NIC), transform</li>
| |
| - | <li>redo fluorescence experiment using the new cultures as well - report fluorescence per OD.
| |
| - | do it in replicate, also use minimal media</li>
| |
| - | <li>Do I751250(in pDAWN) + T9002 (in psb1a3) co-transformation</li>
| |
| - | <li>MoveT9002 into psb1k3 to be compatible with pdawn - first re-transform psb1k3 then digest/ligate/transform/miniprep</li>
| |
| - | </ol></li>
| |
| - |
| |
| - | <li>9-Jul<ol>
| |
| - | <li>Make clear media for lux stuff hold on this until device</li>
| |
| - | <li>Run gel to confirm MsssI PCR assembly/ before meeting-then gel extract the correct band </li>
| |
| - | <li>nanodrop pTetGFPUSER/ TetR USER/ promoters (pcr products) </li>
| |
| - | <li>Run gel to confirm tetR/ 4 promoters PCR</li>
| |
| - | <li>Troubleshoot these USER PCR's (msssI,tetR,4 promoters): msssi</li>
| |
| - | <li>Label gel and send out images and analysis (tetr/promoters)</li>
| |
| - | <li>1st round DBD pcr's add his tag and flex user site</li>
| |
| - | <li>grow up c0040 (TetR) glycerol stock and miniprep and then sequence</li>
| |
| - | <li>redo TetR sequencing of our current miniprep just to be sure</li>
| |
| - | <li>transform C0179 (lasR without LVA degradation tag) in DH5a, pick colonies, miniprep/glycerol stock</li>
| |
| - | <li>miniprep T9002/amp from glyc at H10 and C0079 (grow overnight on Thurs to miniprep at same time as C0179)</li>
| |
| - | <li>Redesign LuxI into pDAWN primers (NdeI, BamHI)</li>
| |
| - | </ol></li></ul></div>
| |
| - | </div>
| |
| - |
| |
| - | <div>
| |
| - | <h2>WEEK 6</h2>
| |
| - | <div class="box"> <h2>July 10 2013 - July 17 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>11-July<ol>
| |
| - | <li>when we have purified pcr USER product of petgfp/tetR/ promoters - USER fuse the reporter plasmids - note: if there is only one band, then you don't even need to pcr purify</li>
| |
| - | <li>Trouble shoot 1st round DBD pcr's add his tag and flex user site</li>
| |
| - | <li>We've grown up c0040 (TetR): make a glycerol stock and miniprep and then sequence-it grew in the wrong culture, currently waiting for it to regrow</li>
| |
| - | <li>redo TetR sequencing of our current miniprep just to be sure</li>
| |
| - | <li>redo 1st round DBD pcr's - add his tag and flex site</li>
| |
| - | <li>miniprep T9002/amp from glyc at H10 and C0079 (grow overnight on Thurs)</li>
| |
| - | <li>Redesign LuxI into pDAWN primers (NdeI, BamHI)</li>
| |
| - | <li>use linearized psb1k3, go ahead and do the digest/ligation t9002 </li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>13-July<ol>
| |
| - | <li>add in primers to MsssI PCR</li>
| |
| - | <li>redo Step 1 of plan - pcr assembly MsssIwith new primers - pfu. do we have enough msssi to bother with taq?</li>
| |
| - | <li>do step 7 simultaneously - ie. take some of Part 1 pot into new reaction pot with (7) primers</li>
| |
| - | <li>PCR purify pfu version of 1st round DBD pcr</li>
| |
| - | <li>step 3 - 2nd Round dbd PCR </li>
| |
| - | <li>Step 5 of plan - PCR pet26b (50ul rxn) (ie. go ahead with zf/tale even though we need to wait up for cas9 backbone)</li>
| |
| - | <li>streak lawned user fusions of reporter plasmid, grow in SOC for an hour, dilute and use new plates to get single colonies</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>14-July<ol>
| |
| - | <li>trouble shoot gels of step (4) and (3) and (5). figure out which PCR to redo. which to go ahead and digest etc</li>
| |
| - | <li>run tae gel with both msssi pcrs, re-run pet26b pcr, re-run cas9rd2, re-run sgRNA1 pcr, run all of TALE1rd2 for gel extraction</li>
| |
| - | <li>image/analyze big TAE gel. Gel extract TALE 2.5kb</li>
| |
| - | <li>step (5) - redo pet26b PCR with lower annealing temp, troubleshoot more</li>
| |
| - | <li>step (3) - redo cas and TALE PCR for cleaner result. troubleshoot somehow.</li>
| |
| - | <li>Gel image redo of cas, Tale, sgrna1, and pet26b. </li>
| |
| - | <li>step 4 - redo sgRNA1 PCR, maybe lower annealing temp?</li>
| |
| - | <li>Digest 44251 with EcoRI and XbaI</li>
| |
| - | <li>Gel extract 44251 digest</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>15-July<ol>
| |
| - | <li>3rd try cas9/TALE rd2 and pET26b PCR.</li>
| |
| - | <li>salvage/Redo MsssI pcr</li>
| |
| - | <li>redo TetR PCR from biobrick and from Mo's new miniprep</li>
| |
| - | <li>grow up pet26b glycerol stock for miniprep</li>
| |
| - | <li>Write Protocol: Check rep plasmids fluorescence, if there are no NIC colonies: pick 3 colonies of each to grow,</li>
| |
| - | <li> Rep Plasmids: take dilutions and see if they fluoresce with tetracycine induction, use this to choose clones for glycerol stock, miniprep, and seq. </li>
| |
| - | <li>grow up R0071 colony for miniprep</li>
| |
| - | <li>redo the standard curve - measure tomorrow</li>
| |
| - | <li>test sender receiver in M9 - growing up, need to induce tomorrow (maybe)</li>
| |
| - | <li>redo testing spent media - growing up, need to induce tomorrow </li>
| |
| - | <li>troubleshoot kan tranformations - WE CANT LET THE KANAMYCIN WIN!</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>16-July<ol>
| |
| - | <li>run Gel of Cas/Tale rd 2 & pET26b</li>
| |
| - | <li>Miniprep pet26b then continue with step (4)</li>
| |
| - | <li>step4- digest/ligate (NIC)/ transform sgrna1 w/ pet26b. do simultaneously with (7)</li>
| |
| - | <li>Check RP's colonies fluorescence/ NIC colonies, pick 5 to grow.</li>
| |
| - | <li>Since NIC (no insert control) grew, we need to colony PCR these guys</li>
| |
| - | <li> Order internal primers for MsssI</li>
| |
| - | <li>Redo Cas9 and Tale rd 2 with PFU turbo cx </li>
| |
| - | <li>do we need new reverse primers for Cas9 and Tale rd 2?</li>
| |
| - | <li>Figure out western/SDS page to see if fusions are being expressed - send protocol. Note we have his-tagged our fusions, if that helps at all</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>17-July<ol>
| |
| - | <li>run pET26b gel</li>
| |
| - | <li>continue (4) - colony pcr, grow 3 correct cultures (pet26b+sgrna)</li>
| |
| - | <li>Check Rep. Plasmid's fluorescence, pick 3 colonies for colony PCR if NIC is empty, 5 if NIC is small growth, start over if NIC grows well</li>
| |
| - | <li>Plan tetracycline induction expt</li>
| |
| - | <li>grow up T9002 chlor for miniprep</li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 7</h2>
| |
| - | <div class="box"> <h2>July 18 2013 - July 24 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>18-July<ol>
| |
| - | <li>Redo pET26b USER PCR with pfu</li>
| |
| - | <li>Call taq/Pfu company about 5.5 kb amplicon</li>
| |
| - | <li>redo pTetGFP w taq/ Pfu</li>
| |
| - | <li>Miniprep pet26b+sgRNA1 and send for sequencing with primers from positions E1 and E2</li>
| |
| - | <li>Save pET26b+USER pcr product in 1.5 ml in misc box. Save 2 best pTetGFP+USER pcr products in 1.5ml in misc box. type up PCR protocol and send (we have to repeat this PCR for the modified pET26b+sgRNA1 now) </li>
| |
| - | <li> USER Fuse new pTetGFP+TetR+5varpromoters and transform</li>
| |
| - | <li>Get sgRNA in psb1A3 sequenced - use vf vr</li>
| |
| - | <li>do co transformation of dcas and sgrna in psb1a3 </li>
| |
| - | <li>Call NEB about M.Sssi</li>
| |
| - | <li>Grow up T9002 in chlor</li>
| |
| - | <li>Miniprep T9002 in chlor</li>
| |
| - | <li>co transform T9002 in chlor and I751250 in AMP - waiting on t9002 miniprep</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>19-July<ol>
| |
| - | <li>MsssI PCRs with new internal primers</li>
| |
| - | <li>redo cas9/tale with 7.17 new (F) and (R) and pfu</li>
| |
| - | <li>skype with Stef @ 3:00</li>
| |
| - | <li>Order seq primers for reporter plasmids</li>
| |
| - | <li>run C2,T2,Z2 gel (thermocycler count dracula)</li>
| |
| - | <li>mp dcas, dcas-sgrna</li>
| |
| - | <li>Design primers for M.SssI from NEB - waiting on dude from NEB on sequence</li>
| |
| - | <li>PCR M.SssI from NEB - waiting on primers</li>
| |
| - | <li>PCR luxI out of v. fischeri, digest it and ligate it into pDAWN then transform ----- waiting for primers</li>
| |
| - | <li>tranform rhl system (c0070, c0071, r0071)</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>22-July<ol>
| |
| - | <li>grow NEB mSssI in chlor/kan,kan,chlor, and no AB LB culture tubes</li>
| |
| - | <li>Re-transform ZF fusion, pET26b-MsssI ligation</li>
| |
| - | <li>Order seq primers for fusion plasmids</li>
| |
| - | <li>Set up time to work out bisulfite seq primers with chris asap</li>
| |
| - | <li>Redo Cas9 and Tale Round 2 PCR</li>
| |
| - | <li> check on pET26b/sgRNA sequencing</li>
| |
| - | <li>digest, gel extract digested fragments of pEt26b and sgRNA1</li>
| |
| - | <li>grow up pTETGFP for positive control for fluorescence expt</li>
| |
| - | <li>grow up pET26b to replenish miniprep stock</li>
| |
| - | <li>grow up r0071 (amp)</li>
| |
| - | <li>digest T9002 and pSB1K3, gel extract</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>23-July<ol>
| |
| - | <li>MPs: first, glycerol stock MsssI, pBAD3, R0071. then miniprep all 3 and pET26b. LIMS. Check ~7:00</li>
| |
| - | <li>Miniprep/glycerol stock/send for seq: pBAD reporter plasmid. NOTE: aliquot some off first for fluorescence induction experiment. (dilute back to .05 and induce at 0.1)</li>
| |
| - | <li>Miniprep pET26b to replace miniprep in spot D9</li>
| |
| - | <li>Check on reporter plasmid re-transformations. Troubleshoot. Re-do transformations as necessary</li>
| |
| - | <li>minprep/glycy/make competent NEB M.SssI cells depending on DC's results</li>
| |
| - | <li> Troubleshoot gel extraction w Spence. Re-do digestion/gel extraction of pET26b and sgRNA1.</li>
| |
| - | <li>ligate, transform pET26b and sgRNA</li>
| |
| - | <li>Colony PCR ZF fusion plasmid (1 PET seq primer, 1 fusion primer) and MsssI-pet26b plasmid (1 pet seq primer, 1 MsssI primer) then grow up for miniprep/seq/glycerol stocking</li>
| |
| - | <li>Digest verify pBAD miniprep</li>
| |
| - | <li>Transform pBAD miniprep into DF's newly competent MsssI strain (use amp + resistance that worked best last night). and test transformation to see that competent cells work ok (psb1a3)</li>
| |
| - | <li>Order Primers for COBRA</li>
| |
| - | <li>Refine ATC induction protocol with Spencer</li>
| |
| - | <li>inoculate M9 cultures with pBAD and with pTetGFP and begin tetracycline/ATC induction experiment. compelling data</li>
| |
| - | <li>Watch SAAST demo of SDS Page ~ 1:30pm</li>
| |
| - | <li>Transform last night's USER reporter plasmids </li>
| |
| - | <li>Digest/Ligate/Transform pET26b with MsssI</li>
| |
| - | <li>LIMS minipreps</li>
| |
| - | <li>Grow up MsssI in correct antibiotics</li>
| |
| - | <li>grow up more T9002 in chlor from glycyrol stock</li>
| |
| - | <li>get T9002 from Brad (or re-digest), ligate reporter plasmid, transform</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>24-July<ol>
| |
| - | <li>Miniprep ZF fusion plasmid , glycerol stock, send for sequencing</li>
| |
| - | <li>Miniprep pET26b alongside</li>
| |
| - | <li>Send pBAD3 reporter plasmid for sequencing (4 primers)</li>
| |
| - | <li>send minipreps of 44249,27969, and 12612 from positions A2, G5, C10 for complete sequence verification ASAP. These are the exact minipreps used for our fusion plasmids- we ran out of the dCas miniprep, so we need more of that before it can be sentin for sequencing</li>
| |
| - | <li>Transform MsssI(+) kan and its NIC - these are in Attila the HUn. Ligations, so use 3 uL and plate all of it undiluted</li>
| |
| - | <li>Transform your redo of pET26b+sgRNA1, and NIC. plate all, undiluted </li>
| |
| - | <li>grow up more pET26b</li>
| |
| - | <li>Continue taking time points of ATC induction (ie 16 hours etc)</li>
| |
| - | <li>Redo TALE round 1, then redo TALEround 2</li>
| |
| - | <li>More colony PCR of ZF fusion plasmid to try to get more clones</li>
| |
| - | <li>Pick Rep Plas colonies for colony PCR/ grow</li>
| |
| - | <li>Re-do digestion, gel extraction, ligation of pET26b+sgRNA1 and NIC</li>
| |
| - | <li>If you have more sample - re run meth diagnostic in 1.5% TBE Gel (load all!). </li>
| |
| - | <li>grow up dcas9 44249 for miniprep</li>
| |
| - | <li>test pBAD reporter with arabinose </li>
| |
| - | <li>SDS page the zfp</li>
| |
| - | <li> order miniprep columns</li>
| |
| - | <li>grow up zf 4, 11 in kan. grow up pLci 10 in amp</li>
| |
| - | <li>grow up pdawn</li>
| |
| - | <li>miniprep T9002 in chlor</li>
| |
| - | <li>redo digestion, gel extraction, ligation of T9002 and pSB1K3 and NIC</li>
| |
| - | <li>redo transformation of T9002/pSB1K3 and NIC</li>
| |
| - | <li>Take reading of sender experiment @ 7</li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 8</h2>
| |
| - | <div class="box"> <h2>July 25 2013 - July 31 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>25-July<ol>
| |
| - | <li>First thing, miniprep glyc stock ZF Fusion plasmid for better yield and ASAP send out for seq again so we can have seq before meeting</li>
| |
| - | <li>miniprep dcas9 44249</li>
| |
| - | <li>Miniprep/ glycerol stock ZFP4, ZFP11, and pLcI10. send for sequencing (fusion plasmids and rep plasmid)</li>
| |
| - | <li>Send pBAD3 reporter plasmid for sequencing (4 primers)</li>
| |
| - | <li>Transform Reporter plasmid ligations (pcr tubes in freezer) (6)</li>
| |
| - | <li> Transform 3 ZF fusion plasmid into BL21 for protein expression and SDS page, also cotransform with pBAD3 </li>
| |
| - | <li>PCR purify sgRNA PCR product, digest, gel extract, ligate to pET26b, transform again</li>
| |
| - | <li>Redo Pet26b + MsssI Digestion / ligation with new NdeI</li>
| |
| - | <li>figures from atc induction</li>
| |
| - | <li>Redo dcas round 1, then redo dcas round 2</li>
| |
| - | <li>confirm/order BL21 (DE3) cells ~ 7:20</li>
| |
| - | <li>Design primers to amplify dCas9 in one round. design first round primers for phusion polymerase. </li>
| |
| - | <li>check if other methylation sensitive enyzmes are in target sequence</li>
| |
| - | <li>Design primers to add BstUI site before promoter in reporter (site directed mutagenesis)</li>
| |
| - | <li>User fuse and transform new TALE with msssi and pet26b</li>
| |
| - | <li>Repeat ATC induction experiment @ 1 time point (media, ptet, reporter induced at 0-1280)</li>
| |
| - | <li>BstUI assay/verification for pLcI 10 </li>
| |
| - | <li>try dCas9 in one round with new primers when they come in</li>
| |
| - | <li>transform ab's pet26 b msssi ligation and NIC marked L in the fridge</li>
| |
| - | <li>Transform Barry Canton's part</li>
| |
| - | <li>redo AHL experiment with new AHL </li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>29-July<ol>
| |
| - | <li>colony PCR on cultures of pet26b+sgrna & NIC using (R)sgRNA1-XhoI from H2 and pet26b(+) Fwd seq from E2 = exp band 414bp</li>
| |
| - | <li>glycerol stock ZF fusion 11 in BL21 and dilute and keep culture going to use for protein expression and SDS page</li>
| |
| - | <li>colony PCR on cultures 5 reporter plasmids & NIC with primers VF2 and (R) RepPlasSeq1 from i9 = exp band 1650bp</li>
| |
| - | <li>Colony PCR on TALE Fusion plasmid with pET26b(+) Fwd seq from E2 and (R)MsssI2 from H8= exp band 3.5kb</li>
| |
| - | <li>Glycerol stock / Miniprep the verified cultures/glycerol stock / miniprep I13202 (Canton's sender)</li>
| |
| - | <li>PCR user ends onto pet26b+sgRNA1 </li>
| |
| - | <li>redo colony PCR for tale, pLac, pDNAa (run gel Tuesday morning then grow up successful colonies ASAP)</li>
| |
| - | <li>grow up sender/receiver cotransformation </li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>30-July<ol>
| |
| - | <li>AM: Run Gel on Colony PCRS</li>
| |
| - | <li>2pm: Grow cultures of 5 biobricks, NEB</li>
| |
| - | <li>Dilute to .1, then induce with aTc</li>
| |
| - | <li>Figure out what went wrong with TetR Sequencing - fix map</li>
| |
| - | <li>BEFORE 5PM: Send new reporters, TALE, pet26b+sgrna for sequencing</li>
| |
| - | <li> AM : Run Gel on pet26b+sgRNA1 PCR </li>
| |
| - | <li>USER fuse and transfrom dCas9+msssi+pet26b+sgRNA, and its (NIC) - ask spencer for cannons</li>
| |
| - | <li>Write protocol for protein expression based on spencers</li>
| |
| - | <li>Before 5 pm: Acquire from Chow Lab / Cell center all materials for Protein Expression and SDS PAGE</li>
| |
| - | <li>minprep pBAD3 (3:30 PM Tuesday)</li>
| |
| - | <li>run gels of col pcr of TALE and MsssI, grow up the right clones</li>
| |
| - | <li>update lims with seq results; send out type up</li>
| |
| - | <li>co-transform zinc finger w pbad3 - commercial bl21. consider doing single transformations on double antibiotic plates as controls</li>
| |
| - | <li>transform MsssI ligation and NIC. Transform NEB MsssI - dh5@ max eff</li>
| |
| - | <li>try canton sender's media with receiver</li>
| |
| - | <li>start cultures in AM</li>
| |
| - | <li> Gantt Chart</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>31-July<ol>
| |
| - | <li>glycerol stock and miniprep k325259, k325219, k577893, k145279</li>
| |
| - | <li>re-do MsssI colony PCR - there were no bands</li>
| |
| - | <li>grow up culture of NEB cells</li>
| |
| - | <li>look into choosing McrA-, McrB- cells : figure out whats up with NEB's cells</li>
| |
| - | <li>Co-transform ZFP11 with pBAD1 in BL21 ~500ng each. Transform just ZFP11 and just pBAD1 in BL21. Transform dcas9 fusions and NIC in DH5@max. Transform NEB Plasmid in DH5@max. </li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 9</h2>
| |
| - | <div class="box"> <h2>Aug 1 2013 - Aug 7 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>4-Aug<ol>
| |
| - | <li>glycerol stock and miniprep k206500 and Tale18+pBAD1 BL21 cotransformation</li>
| |
| - | <li>send out co trans growth curve </li>
| |
| - | <li>Induction and SDS PAGE on ZF11, TALE 18, and MsssIPET12</li>
| |
| - | <li>-80c lims missing coordinates</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>5-Aug<ol>
| |
| - | <li>Induce M12,T + B1,L4 co trans with .1mM</li>
| |
| - | <li>miniprep MsssiPet12Dh5a, ZF11dh5a, pLci4NEb10, TALE18Dh5a</li>
| |
| - | <li>Grow up Glycerol Stocks of NEB+pBad1, NEB+pLcI4 for induction Experiment</li>
| |
| - | <li>ZF11+pBad1 in DH5@ - use 15 uL</li>
| |
| - | <li>ZF11+pBad1 in T7 express - use 20 uL</li>
| |
| - | <li>ZF11+pLcI4 in DH5@</li>
| |
| - | <li>ZF11+PLCI4 IN T7 express</li>
| |
| - | <li>NEB MsssI + PLcI4 in ER1821 (small tubes in tip box, tape label on top of box - use 50uL)</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>7-Aug<ol>
| |
| - | <li>Look through the miniprep boxes and see which minipreps are relevant to the project and have <10uL left, innoculate fresh cultures of those for miniprepping tomorrow</li>
| |
| - | <li>BsoBI - methylation insensitve isochizmer of avaI</li>
| |
| - | <li>order more cpg methylase</li>
| |
| - | <li>Design gibson dcas9 primers</li>
| |
| - | <li>make alliquots of K, A and C</li>
| |
| - | <li>Make plate LIMS </li>
| |
| - | <li>project abstract</li>
| |
| - | <li>co transform TALE 18 + pbad1 in T7 express, TALE18+pLcI4 in T7 Express, TALE18+pBad1 in DH5a, TALE18+pLcI4 in DH5a, ZF11+pBad1 in DH5a, ZF11+pLcI4 in DH5a</li>
| |
| - | <li>grow up NEB+pbad1 and NEB+pLcI4 for induction experiment</li>
| |
| - | <li>meet with issadore to talk about new device</li>
| |
| - | <li>work on iterating the device</li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 10</h2>
| |
| - | <div class="box"> <h2>Aug 8 2013 - Aug 14 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>8-Aug<ol>
| |
| - | <li>Do t7 induction and methylation sensitive digest for zinc finger co-trans</li>
| |
| - | <li>Induce NEBMsssI with reporters for methylation sensitive digest</li>
| |
| - | <li>Prepare presentation for Orkan and Thuy</li>
| |
| - | <li>Do minipreps of: pBad1, NEBMsssI in the fridge</li>
| |
| - | <li>LIMS all the minipreps in random racks in the freezer</li>
| |
| - | <li> pick colonies from yesterday's cotransformations</li>
| |
| - | <li>transform zif 11 + pbad1 in dh5@</li>
| |
| - | <li>transform zif 11 + plci4 in dh5@</li>
| |
| - | <li>transform for methylation repression screen: k584000, k774007, j04450, k079050</li>
| |
| - | <li>design primers to gibson assemble luxi into pdawn</li>
| |
| - | <li>ligate and transform t9002 in psb1ak3</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>9-Aug<ol>
| |
| - | <li>send tale18 for re-do sequencing</li>
| |
| - | <li>induce TALE co trans for digestion assay. choose pLcI4 or Pbad1 and bstui or avaI respectively</li>
| |
| - | <li>transform TALE18, ZF11 in T7 express to grow up tomorrow for SDS Page, glycerol stock, and miniprep</li>
| |
| - | <li>redo zinc finger gel, linearizing, </li>
| |
| - | <li>CUT QUORUM SENSING PROJECT</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>12-Aug<ol>
| |
| - | <li>Miniprep TALE4 , new fusion clone, and send for sequencing asap</li>
| |
| - | <li>Image and send out gel</li>
| |
| - | <li>get mikes compatible GFP vector gorw it up</li>
| |
| - | <li>Send out notes from JMIL</li>
| |
| - | <li> Glycerol stock ZF11 T7, TALE 18 T7 express</li>
| |
| - | <li>Is it possible the co-trans work in BL21 and not in T7 express or dh5a?-- T7express is a BL21 derivative, no reason cotrans should work in one over the other, we're moving it all to one plasmid anyway </li>
| |
| - | <li>make more LB</li>
| |
| - | <li>Miniprep ZF11 T7, TALE 18 T7 express</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>13-Aug<ol>
| |
| - | <li>verify order of (R)MsssIGA2</li>
| |
| - | <li>type up cas9 assembly protocol and go over</li>
| |
| - | <li>send chris biseq primers</li>
| |
| - | <li>Design way to add target to TALE and ZF and MsssI</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>14-Aug<ol>
| |
| - | <li>Send out report on zinc finger linker length, if we can perfectly reproduce it or not.</li>
| |
| - | <li>Learn assembly protocol from JT</li>
| |
| - | <li>Send chow Timeline and what expts, by when, by whom, resource allocation</li>
| |
| - | <li>Incorporate Chow and others suggestions into the powerpoint</li>
| |
| - | <li>need to check TALE binding sequence</li>
| |
| - | <li>SDS page </li>
| |
| - | <li>check which items we need for future protocols</li>
| |
| - | <li>talk to chris about 'perfect primers'</li>
| |
| - | <li>move glycerol stocks in row F of 2012 box to a 2013 box, re-LIMS</li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 11</h2>
| |
| - | <div class="box"> <h2>Aug 15 2013 - Aug 21 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>15-Aug<ol>
| |
| - | <li>at 4: grow up addgene dcas9, pET26b+sgRNA lig (1 or 2) for miniprepping</li>
| |
| - | <li>phosphorylate and anneal target oligos for dcas</li>
| |
| - | <li>miniprep and glycerol stock pET26b in T7 (1 and 2), INBNC-mCherry in BL21 (k), intein-mCherry in BL21(amp)</li>
| |
| - | <li>digest pET26b with NotI and XhoI, column purify (3X)</li>
| |
| - | <li>digest pET26b+sgRNA1 with BamHI and XhoI, column purify</li>
| |
| - | <li>ligate target oligos with pET26b/sgRNA backbone (for cas plasmid)</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>16-Aug<ol>
| |
| - | <li>miniprep dcas9, pET26b+sgRNA</li>
| |
| - | <li>phosphorylate/ligate target oligos with pET26b backbone for ZFN/TALE/M.SssI</li>
| |
| - | <li>transform all ligations (TALE/ZFNM.SssI Target, Cas9 Target) in pET-26B</li>
| |
| - | <li>Sort Purchase spreadsheet for Brian</li>
| |
| - | <li>Use Brian's tips to refine the intro slides from the powerpoint</li>
| |
| - | <li>Make detailed plan of when we are spending money, why we are spending it, and how much of it we will be spending</li>
| |
| - | <li>make detailed plan of what figures we need (once plasmids are cloned), what assays to perform</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>18-Aug<ol>
| |
| - | <li>glycerol stock and miniprep backbones with target sequence</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>19-Aug<ol>
| |
| - | <li>PCR sequenced zinc finger fusion, M.SssI, tale fusion (so XbaI and NotI can be used to digest/ligate onto backbone) using primers (F) OnePlasInsert and (R) OnePlasInsert</li>
| |
| - | <li>PCR M.SssI template that DC Gibson assembled with (F) M.SssIGA2 and (R) M.SssIGA3 - see note on JT's dcas protocol about this reverse primer</li>
| |
| - | <li>PCR Cas9 from Addgene plasmid</li>
| |
| - | <li>digest pET26b+target backbone with XbaI and NotI, gel extract</li>
| |
| - | <li>digest pET26b/sgRNA+target backbone with NcoI and NdeI, gel extract</li>
| |
| - | <li>send out new and improved video plan </li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>20-Aug<ol>
| |
| - | <li>digest zf, tale, M.SssI PCR products with XbaI and NotI, column purify</li>
| |
| - | <li>order new bisulfite seq primers</li>
| |
| - | <li>work on modularity of one plasmid system</li>
| |
| - | <li>FILM DAY</li>
| |
| - | <li>miniprep dcas and zfn target backbones, elute with water instead of buffer</li>
| |
| - | <li> How else can we clone MsssI into pET26b+Target? (order primers)</li>
| |
| - | <li>ligate pET26b+target (after digestion) with zf, tale PCR products (after digestion); remember the no-insert control</li>
| |
| - | <li>transform pET26b+target + zf, tale ligations into DH5a</li>
| |
| - | <li>Gibson assemble the digested pET26b/sgRNA+target backbone (in-progress B4, conc 12.0) with the PCR'ed cas9 and PCR'ed M.SssI that DC Gibson assembled; call NEB for instructions on how to do this</li>
| |
| - | <li>transform the Gibson assembly into DH5a</li>
| |
| - | <li>repeat assay of backbones+target with AvaI and BglII, using new minipreps, in vitro methylation time course</li>
| |
| - | <li>confirm pet26b+Target lig5 (aka zfn target lig 5) by re-doing the col pcr on both this miniprep and normal pet26b miniprep and running only a small amount on a 2% TBE gel to get distinct bands with 30 bp difference</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>21-Aug<ol>
| |
| - | <li>checking tale and ZF PCR, then digest with XbaI and NotI-HF with cutsmart, ligate to pET26b/target backbone (in-progress box B5, 11 ng/uL) then transform based on typed protocol</li>
| |
| - | <li>gibson assemble and transform CAS plasmid, using backbone pET26b/sgRNA/target (in-progress B4, conc 12.0) </li>
| |
| - | <li>redo miniprep of target+backbones and do methylation test on pet26b+sgran+target. Methylation asay in progress</li>
| |
| - | <li>PCR MsssI, run Gel, Digest with NdeI and NotI, gel extract the larger band</li>
| |
| - | <li>bk is running gel for MsssI PCR</li>
| |
| - | <li>redo Target in Pet26b ligation and transformation </li>
| |
| - | <li>write updated budget for brian</li>
| |
| - | <li>Work on Human Practices Essay</li>
| |
| - | </ol></li>
| |
| - |
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 12</h2>
| |
| - | <div class="box"> <h2>Aug 22 2013 - Aug 28 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>22-Aug<ol>
| |
| - | <li>check for sequencing on pet26b+sgrna+target so we can go ahead and gibson assemble and transform with cas-msssI</li>
| |
| - | <li>gibson assemble and transform CAS plasmid, using backbone pET26b/sgRNA/target (in-progress B4, conc 12.0) </li>
| |
| - | <li>Do minipreps, digest methylation time course with xbai and avai, do methylation assay with controls</li>
| |
| - | <li>Digest MsssI pcr producrt with ndei and noti, gel extract the larger band</li>
| |
| - | <li>Digest pet26b+target with ndei and noti for msssi ligation, gel extract larger band</li>
| |
| - | <li>make fresh buffer PE </li>
| |
| - | <li>re-do making pet26b+target; look up if overhangs are stable and decide about using backbone digest or re-doing digest and then ligate and transform</li>
| |
| - | <li>Fundraise for next year's team</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>23-Aug<ol>
| |
| - | <li>check for sequencing on pet26b+sgrna+target so we can go ahead and gibson assemble and transform with cas-msssI</li>
| |
| - | <li>gibson assemble (in-progress C7) and transform CAS plasmid into NEB10</li>
| |
| - | <li>Send out methylation time course results</li>
| |
| - | <li>Digest MsssI pcr producrt with ndei and notiHF, gel extract the larger band</li>
| |
| - | <li>bisfulite primers = look at virtual gels, Go over with spencer or mike in AM, send for approval</li>
| |
| - | <li>re-arrange the inprogress box</li>
| |
| - | <li>Digest pet26b+target with ndei and noti for msssi ligation, gel extract larger band</li>
| |
| - | <li>colony PCR on pet26b+target with new (F)Target2Primer and pet26brevseq, include pet26b as control</li>
| |
| - | <li>prepare finalized plasmid maps - PST backbone. cas with his tag from (R)MsssIGA2</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>24-Aug<ol>
| |
| - | <li>colony PCR the Gibson assembled cas plasmid, grow up colonies - no colonies :\</li>
| |
| - | <li>miniprep pet26bsgrnaTarget#3 and tell josh the concentration</li>
| |
| - | <li>ligate MsssI and PST3, transform</li>
| |
| - | <li>miniprep TALE and ZF</li>
| |
| - | <li>Digest TALE, ZF and backbone pet26b+sgRNA1+Target ligation 3 (aka PST3) with NcoIHF and XbaI. Gel extract, ligate, transform</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>25-Aug<ol>
| |
| - | <li>gibson assemble (in-progress C7) and transform CAS plasmid into NEB10</li>
| |
| - | <li>Miniprep Pet26b+sgrna+target 3 (PST3) - borrow column or save culture in fridge for Miniprep tomorrow</li>
| |
| - | <li>digest PST3 with NcoIHF and XbaI, gel extract</li>
| |
| - | <li>Ligate your PST3 with Tale, and with ZF, and NIC, and transform into NEB10</li>
| |
| - | <li>Digest PST3 with NdeI and NcoIHF, column purify</li>
| |
| - | <li>Ligate this PST3 with MsssI in progress box, and NIC, and transform </li>
| |
| - | <li>Order bisulfite primers for ON target</li>
| |
| - | <li>redo outro voice over</li>
| |
| - | <li>prepare "notebook" pages for website</li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 13</h2>
| |
| - | <div class="box"> <h2>Aug 29 2013 - Sep 4 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>1-Sep<ol>
| |
| - | <li>COBRA for ON target pilot attempt - methylated and unmethylated</li>
| |
| - | <li>Order BiSeq primers for OFF target </li>
| |
| - | <li>replate T7 transformations of ZF and TALE clones</li>
| |
| - | <li>Transform your overnight ligation mix of MsssI and NIC, plate all on full plates</li>
| |
| - | <li>Prepare ZF and TALE sequencing of all clones for early submission Tuesday AM</li>
| |
| - | <li> Gibson assemble Cas plasmid</li>
| |
| - | <li>Order primers for cloning msssi biobricks</li>
| |
| - | <li>Submit reporter biobricks</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>2-Sep<ol>
| |
| - | <li>Col PCR MsssI and Gibson Assembly</li>
| |
| - | <li>COBRA for ON target pilot attempt - methylated and unmethylated</li>
| |
| - | <li>need to get TaqåI enzyme for COBRA @ target site. waiting response from NEB</li>
| |
| - | <li>write up cobra workflow</li>
| |
| - | <li>get brian to sign travel grant</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>3-Sep<ol>
| |
| - | <li>col pcr gibson assembly</li>
| |
| - | <li>send Cas clone1 for seq</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>4-Sep<ol>
| |
| - | <li>Clone msssi biobrick with your primers</li>
| |
| - | <li>clone msssi in pst3 backbone</li>
| |
| - | <li>methylation time course - GELS FOR PENN APPS</li>
| |
| - | <li>attempt digestion of tale , zf clones</li>
| |
| - | <li>zf target site missing?</li>
| |
| - | <li>do COBRA with taqI enzyme and XhoI enzyme </li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 14</h2>
| |
| - | <div class="box"> <h2>Sep 5 2013 - Sep 11 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>5-Sep<ol>
| |
| - | <li>poster presentation 5-630 bodek lounge tues 9/10; meeting with brian next tues 8am?; waiting to hear from orkan and david gdula.</li>
| |
| - | <li>consider alt tale binding sites implications for meth digest</li>
| |
| - | <li>test OFF target cobra primers</li>
| |
| - | <li>call NEB about bisulfite converting plasmid</li>
| |
| - | <li>send fusions for seq</li>
| |
| - | <li>col pcr on biobricks </li>
| |
| - | <li>cas col pcr with proper pos ctrl</li>
| |
| - | <li>essay for bioethics journal</li>
| |
| - | <li>prep interview questions</li>
| |
| - | <li>Mack Institute blurb</li>
| |
| - | <li>travel grant proposal</li>
| |
| - | </ol></li>
| |
| - | </br>
| |
| - | <li>6-Sep<ol>
| |
| - | <li>look at danny's triplicate gels and figure out what s what and redo the graphs</li>
| |
| - | <li>run the gel</li>
| |
| - | <li>call neb and pick up the thing from chris</li>
| |
| - | <li>CAS</li>
| |
| - | <li>use phosphatase on msssi ligation</li>
| |
| - | <li>cobra- digest off target site and get bisulfite kit from chris to see if we can get more complete bisulfite data</li>
| |
| - | <li>order columns/xbaI</li>
| |
| - | <li>wiki</li>
| |
| - | <li>work on magellin</li>
| |
| - | <li>Compare ON primers 3&6 with ON 1&5 for Cobra</li>
| |
| - | <li>PCR OFF target with 3&7 in 50uL for purification and digestion</li>
| |
| - | <li>Digest OFF from 3&7 with TaqI and BamHIHF</li>
| |
| - | <li>presentation for chow</li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 15</h2>
| |
| - | <div class="box"> <h2>Sep 12 2013 - Sep 18 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
| |
| - |
| |
| - | <li>18-Sep<ol>
| |
| - | <li>write up websites for biobricks, grow up cultures</li>
| |
| - | <li>miniprep biobricks (psb1c3 msssI's and reporters) and go to postoffice</li>
| |
| - | <li>Digest out RFP (wonders f10); gel extract it **alongside gel fragment of the backbone In Progress F1** and ligate to PST BB backbone</li>
| |
| - | <li>Digest MsssI and MsssILinker (in progress box) and ligate to PST BB backbone</li>
| |
| - | <li>Col PCR new ZF target with (F)Target2Primer *note exact primer name</li>
| |
| - | <li>Digest and ligate new ZF target backbone with ZF fusion </li>
| |
| - | <li>Digest and Ligate PST3 with MsssI</li>
| |
| - | <li>Run gradient on biseq primers</li>
| |
| - | <li>Work with MO- who is digesting MsssI and MsssI linker (in progress box) and ligate to PSB1C3 because that colony pcr was sketchy</li>
| |
| - | <li>animation</li>
| |
| - | <li>Essay</li>
| |
| - | </ol></li>
| |
| - | </div>
| |
| - | </div>
| |
| - | <div>
| |
| - |
| |
| - | <h2>WEEK 16</h2>
| |
| - | <div class="box"> <h2>Sep 19 2013 - Sep 25 2013</h2>
| |
| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
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| - |
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| - | <li>19-Sep<ol>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li> </li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | </ol></li>
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| - | </br>
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| - | <li>25-Sep<ol>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | </ol></li>
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| - | </div>
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| - | </div>
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| - | <div>
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| - |
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| - | <h2>WEEK 17</h2>
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| - | <div class="box"> <h2>Sep 26 2013 - Oct 2 2013</h2>
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| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
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| - |
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| - | <li>26-Sep<ol>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li> </li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | </ol></li>
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| - | </br>
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| - | <li>2-Oct<ol>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | </ol></li>
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| - | </div>
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| - | </div>
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| - | <div>
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| - |
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| - | <h2>WEEK 18</h2>
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| - | <div class="box"> <h2>Oct 3 2013 - Oct 9 2013</h2>
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| - | <img class="image" src="https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg">
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| - |
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| - | <li>3-Oct<ol>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li> </li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | </ol></li>
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| - | </br>
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| - | <li>6-Oct<ol>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | <li></li>
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| - | </ol></li>
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| - |
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| - | </div>
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| - | </div>
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| - | <div>
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| - |
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| - | <!--to start a new section follow this format and add "week n" to the ul li ( it's before most of the content)
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| - |
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| - | <div>
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| - | <h2>WEEK </h2>
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| - | <div class="box"> <h2>date 1 - date 2</h2>
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| - | <img class="image" src="image url">
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| - |
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| - | <ul><li>date 1<ol>
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| - | <li>something</li>
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| - | <li>something</li>
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| - | <li>something</li>
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| - | </ol></li>
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| - |
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| - | <ul><li>date 2<ol>
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| - | <li>something</li>
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| - | <li>something<ol>
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| - | <li>sub list</li>
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| - | <li>sub list</li></il>
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| - | </li>
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| - | <li>something</li>
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| - | </ol></li>
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| - |
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| - | </div>-->
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| - |
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| - |
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| - | </div></div>
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| - |
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| - |
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| - |
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| - |
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| - |
| |
| - | </body>
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| - | <script>
| |
| - | jQuery(document).ready(function($) {
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| - | var $items = $('#vtab>ul>li');
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| - | $items.click(function() {
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| - | $items.removeClass('selected');
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| - | $(this).addClass('selected');
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| - |
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| - | var index = $items.index($(this));
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| - | $('#vtab>div').hide().eq(index).show();
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| - | }).eq(0).click();
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| - |
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| - | });
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| - | </script>
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| - | </html>
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"
