Team:Penn/Notebook

From 2013.igem.org

(Difference between revisions)
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<head>
<head>
     <title>Notebook</title>
     <title>Notebook</title>
-
       <link href="https://googledrive.com/host/0B4ZBZOYYKBzEVHRaZEdUVGo5cjA" type="text/css" rel="stylesheet"/>
+
       //<link href="https://googledrive.com/host/0B4ZBZOYYKBzEVHRaZEdUVGo5cjA" type="text/css" rel="stylesheet"/>
     <script src="https://googledrive.com/host/0B4ZBZOYYKBzETkFqdnhMeV9fMzA" ></script>
     <script src="https://googledrive.com/host/0B4ZBZOYYKBzETkFqdnhMeV9fMzA" ></script>
     <script src="https://googledrive.com/host/0B4ZBZOYYKBzEZTdBSFdUV19LYjQ" type="text/javascript"></script>
     <script src="https://googledrive.com/host/0B4ZBZOYYKBzEZTdBSFdUV19LYjQ" type="text/javascript"></script>
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     <style>
     <style>
-
          
+
        /************
 +
  *Stylesheet for Penn's iGEM wiki
 +
  *
 +
  **********/
 +
 
 +
/*import the font quicksandlight (using this for > arrow)*/
 +
          @font-face
 +
{
 +
font-family: QuickSandLight;
 +
src: url('https://googledrive.com/host/0B4ZBZOYYKBzEazRybm9PV21QdTA'),
 +
}
 +
 
 +
/*format paragraph, h1, h2, and h3*/
 +
 
 +
         p {
 +
        color: black; /*font color*/
 +
        font-family: arial, sans-serif; /*font is arial, with sans-serif as a back up*/
 +
        font-size: 13px;
 +
        }
 +
 
 +
        h1, h2 {
 +
          color: black; /*font color*/
 +
        font-family: arial, sans-serif;/*font is arial, with sans-serif as a back up*/
 +
        font-size: 15px;
 +
        }
 +
 
 +
        /*h3 will be the same as h1 and h2, but it will be centered*/
 +
        h3 {
 +
        color: black;
 +
        font-family: arial, sans-serif; /*font is arial, with sans-serif as a back up*/
 +
        font-size: 15px;
 +
        text-align: center; /*center*/
 +
 
 +
        }
 +
 
 +
/*links in sidebar*/       
 +
a{
 +
        font-family: arial, sans-serif;
 +
        text-decoration: none; /*gets rid of underlines*/
 +
        color: black;
 +
    }
 +
 
 +
    /*visited link same as regular link*/
 +
    a:visited {
 +
        color: black !important;
 +
        text-decoration: none; /*gets rid of underlines*/
 +
    }
 +
 
 +
    /*link when the mouse hovers over it*/
 +
    a:hover {
 +
        color: #07304b !important; /*font color is dark blue*/
 +
        text-decoration: none; /*gets rid of underlines*/
 +
    }
 +
 
 +
    .navbar ul {
 +
        list-style-type: none; /*gets rid of bullets*/
 +
    }
 +
 
 +
      /*sidebar--fix line heights to get some padding.  change fonts */
 +
        .navbar{
 +
            position: relative;
 +
            list-style-type: none; /*no bullets*/
 +
            line-height: 30px; /*changes line height to give the sidebar links some padding*/
 +
            font-size: 15px;
 +
            font-family: arial, sans-serif;
 +
        }
 +
 
 +
        .navbar li {
 +
        /*not actually sure if this is neccessary, but I'll just leave it here*/
 +
        padding-left: 50px;
 +
        margin-left: -50px;
 +
        }
 +
 
 +
        /*sidebar--change the color of the background of the link on the sidebar*/
 +
        .navbar li:hover {
 +
            width: 100%; /*expand the link to the width of the sidebar.*/
 +
            background-color: rgb(200, 200, 215); /*change background color to light blue*/
 +
        }
 +
 
 +
        /*change color of the link that's active*/
 +
        .active {
 +
            color: #07304b !important;
 +
        }
 +
 
 +
        /*get rid of default list formatting*/
 +
        .dropdown{
 +
            list-style-type: none; /*no bullets or numbering*/
 +
 
 +
        }
 +
        .dropdown-menu{
 +
          list-style-type: none; /*no bullets or numbering*/
 +
}
 +
        /*change the background color of the active link*/
 +
        .dropdown-menu .active {
 +
            background-color: rgb(200,200,215); /*light blue background color*/
 +
            width: 100px !important; /*set the width*/
 +
            padding: 5px;
 +
            margin: 0px !important; /*get rid of margins*/
 +
        }
 +
 
 +
        /*format the arrow that is on the project and about links*/
 +
        .arrow{
 +
            display: inline; /*inline so it stays next to the link*/
 +
            float: right; /*move it to the right*/
 +
            margin-right: 20px; /*but not all the way to the right*/
 +
            font-size: 20px; /*make it bigger*/
 +
            font-family: QuickSandLight, arial, sans-serif; /*quicksand light seems to make the arrows wider looking*/
 +
 
 +
        }
 +
 
 +
        /*format each button on the sidebar drop down menus*/
 +
        .dropdown-menu li {
 +
        background-color: white; /*white background*/
 +
            width: 100px; /*set width*/
 +
        margin: 0px; /*no margins*/
 +
            padding: 5px;
 +
        }
 +
 
 +
/*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
 +
matter of eyeballing.  This is probably not the most efficient way of doing this, but since there's only two dropdowns it works*/
 +
 
 +
/*drop-menu1 is for the project dropdown*/
 +
        #drop-menu1{
 +
            position: absolute; /*absolutely position so it doesn't conflict with other content*/
 +
            left: 155px; /*move it left until it's not in the navbar anymore*/
 +
            top: 27px; /*move it down right next to the project button (higher number is lower on page)*/
 +
            text-align: center; /*center the text*/
 +
            display: inline;
 +
            line-height: 20px; /*give it a big line height so it has some padding*/
 +
            z-index: 9999; /*bring it up front so it's not behind anything*/
 +
            display: none; /*hide it at first*/
 +
        }
 +
 
 +
        /*drop-menu2 is for about (comments are same as above)*/
 +
        #drop-menu2{
 +
            width: 100px;
 +
            position: absolute;
 +
            left: 155px;
 +
            top: 59px; /*move it down next to the about button (higher number is lower on page)*/
 +
            text-align: center;
 +
            display: inline;
 +
            line-height: 20px;
 +
            display: none;
 +
            z-index: 9999;
 +
 
 +
        }
 +
 
 +
        /*wrap the drop-menu to make it a box--this wrap actually is not very important
 +
        I actually am not sure if it still does anything, but I'm leaving it just in case it does*/
 +
        .drop-menu-wrap{
 +
            width: 120px; /*give it a width*/
 +
            background-color: white;
 +
          text-align: center; /*center the text*/
 +
            min-height: 700px; /*give it a minimum height*/
 +
            float: left; /*float it to the left*/
 +
            margin-left: 0px;
 +
        }
 +
 
 +
 
 +
/*format the left wrapper (sidebar).
 +
fixed left is not being used, but I'm keeping it there just in case*/
 +
.left_wrap, .fixed-left {
 +
        margin-left: -15px; /*this makes sure there's no margin between the sidebar and the page*/
 +
        padding-top: 80px; /*padding to the top so the links don't start there*/
 +
            width: 200px; /*give it a width*/
 +
            min-height:  100%; /*height should take up entire height*/
 +
            position: fixed; /*fix it so it stays in place when you scroll*/
 +
            background-color: white;
 +
          /*cross browser opacity*/
 +
        -ms-filter: "progid:DXImageTransform.Microsoft.Alpha(Opacity=80)";
 +
          filter: alpha(opacity=80);
 +
        -moz-opacity: 0.8;
 +
        -khtml-opacity: 0.8;
 +
          opacity: 0.8;
 +
          z-index: 999; /*make sure sidebar is over everything else in case there is some overlap*/       
 +
 
 +
        }
 +
 
 +
 
 +
      /*position the igem logo, resize, and fix it*/
 +
        #igem {
 +
        z-index: 999; /*it should be over everything in case there is overlap*/
 +
        height: 50px; /*fix the height and leave the width at auto*/
 +
            top: 45px; /*position it 45 pixels down*/
 +
            right: 35px; /*and 35 to the right*/
 +
            margin: 30px 15px 15px 15px;
 +
            position: fixed; /*fix it in place so it doesn't move when you scroll*/
 +
            /*give it some transparency (cross browser)*/
 +
            -ms-filter: "progid:DXImageTransform.Microsoft.Alpha(Opacity=80)";
 +
/* IE 5-7 */
 +
  filter: alpha(opacity=80);
 +
  /* Netscape */
 +
  -moz-opacity: 0.8;
 +
  /* Safari 1.x */
 +
  -khtml-opacity: 0.8;
 +
  /* Good browsers */
 +
  opacity: 0.8;
 +
 
 +
        }
 +
 
 +
        /*position the penn logo, resize, and fix it (same comments as above)*/
 +
        #penn {
 +
              z-index: 999;
 +
        height: 40px;
 +
            top: -5px;
 +
            right: 20px;
 +
            margin: 30px 15px 15px 15px;
 +
            position: fixed;
 +
            -ms-filter: "progid:DXImageTransform.Microsoft.Alpha(Opacity=80)";
 +
/* IE 5-7 */
 +
  filter: alpha(opacity=80);
 +
  /* Netscape */
 +
  -moz-opacity: 0.8;
 +
  /* Safari 1.x */
 +
  -khtml-opacity: 0.8;
 +
  /* Good browsers */
 +
  opacity: 0.8;
 +
        }
 +
 
 +
 
 +
      /*******Format background image, padding, margins of background
 +
            ******************/
 +
        .section1 {
 +
            position: relative;
 +
      bottom: 0px;
 +
  padding: 0;
 +
  margin: 0;
 +
  height: 100%;
 +
z-index: -9999;
 +
            margin-left: -15px; /*make sure there's no space between the browser edge and background*/
 +
            padding-left: 200px; /*padding is as big as the sidebar so there's no overlap and it centers without taking that into account*/
 +
            padding-top: 75px; /*give it some padding*/
 +
          overflow: hidden;
 +
        }
 +
 
 +
 
 +
        /*****
 +
        putting the background image in the body instead of section1 to fix gray bar issue
 +
        ******/
 +
        html, body {
 +
            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*/
 +
          /* min-height: 100%; /*make sure it takes up full screen*/
 +
            /*the next few are for cross browser background size.  "cover" makes the image cover the entire container*/
 +
          -webkit-background-size: cover;
 +
          -moz-background-size: cover;
 +
          -o-background-size: cover;
 +
          background-size: cover;
 +
}
 +
 
 +
 
 +
      /*sets margins, padding and width just in case*/ 
 +
    html,body{
 +
        width: 100%;
 +
        margin: 0;
 +
    padding: 0;
 +
 
 +
    }
 +
        /*white text box - margins are auto.
 +
        *Because of padding in the section divs, it should align taking the navbar into account.
 +
        *Use this around content
 +
        */
 +
        .text {
 +
            color: black;
 +
            width: 700px;
 +
            /*the textbox should extend if text goes over this height, but I noticed it doesn't always.
 +
            Hard code the min-height into the specific textbox's html to fix this issue if it occurs*/
 +
            min-height: 500px;
 +
            background: rgb(54, 25, 25); /* Fall-back for browsers that don't
 +
                                    support rgba */
 +
            background: rgba(255, 255, 255, .8);
 +
            margin: auto;
 +
            overflow: hidden;
 +
            padding: 20px;
 +
            margin-top: 20px;
 +
            -moz-border-radius: 10px;
 +
    -webkit-border-radius: 10px;
 +
    -khtml-border-radius: 10px;
 +
            border-radius: 10px;
 +
        }
 +
 
 +
 
 +
      /*get rid of gray bars on top and bottom.  This accounts for some of the leftover wiki formatting*/ 
 +
        #content {
 +
        margin-top: -10px;
 +
        margin-bottom: -20px;
 +
        padding: 0px;
 +
        }
 +
 
 +
 
 +
        /*wrap any number of text columns that add up to less than 750px wide (plus margins), float one left, one right,
 +
        *one or more in the middle (adjust the middle ones' margins until centered or float center if there's just one.
 +
        *The text wrap will keep the margins on either side centered.
 +
        */
 +
        .text-wrap {
 +
 
 +
            overflow: hidden;
 +
            width: 750px;
 +
            margin: auto;
 +
        }
 +
 
 +
        /*give the textboxes for methods a different minimum height and margin*/
 +
        #methods {
 +
            min-height: 50px !important; /*change this if this is to tall/short*/
 +
            margin-bottom: 10px;
 +
        }
 +
 
 +
 
 +
        /*format the title of the sections in the sidebar and turn them 90 degrees*/
 +
        .section-title {
 +
        top: 50% !important;
 +
        left: 2px;
 +
        position: fixed !important;
 +
            font-family: arial, sans-serif;
 +
            font-size: 25px;
 +
            -webkit-transform: rotate(-90deg);
 +
    -moz-transform: rotate(-90deg);
 +
    -o-transform: rotate(-90deg);
 +
    -ms-transform: rotate(-90deg);
 +
    transform: rotate(-90deg);
 +
    z-index: 9999; /*keep it above everything else*/
 +
        }
 +
 
 +
 
 +
 
 +
 
 +
/****************
 +
Getting rid of wiki defaults
 +
*****************/
 +
 
 +
.outer {
 +
width: 100%;
 +
text-align: center;
 +
float: center;
 +
height: 100%;
 +
background-color: white;
 +
}
 +
 
 +
#p-logo {
 +
    position: absolute;
 +
    display: none;
 +
}
 +
 
 +
#content {
 +
width: 100%;
 +
background-color: white;
 +
}
 +
 
 +
#top-section {
 +
    position: absolute;
 +
}
 +
 
 +
.firstHeading {
 +
    position: absolute;
 +
    display: none;
 +
}
 +
 
 +
/*.thumb {
 +
    display: none;
 +
    position: absolute;
 +
}*/
 +
 
 +
 
 +
#search-controls {
 +
display: none;
 +
position: absolute;
 +
}
 +
 
 +
 
 +
#footer {
 +
    display: none;
 +
    position: absolute;
 +
}
 +
 
 +
ul {
 +
    list-style-type: none;
 +
    list-style-image: none;
 +
}
 +
 
 +
.printfooter {
 +
    display: none;
 +
    position: absolute;
 +
}
 +
 
 +
 
 +
.visualClear {
 +
    display: none;
 +
    position: absolute;
 +
}
 +
 
 +
#footer-box {
 +
    display: none;
 +
    position: absolute;
 +
}
 +
 
 +
 
 +
body {
 +
    margin: 10px 0 0 0;
 +
    padding: 0;}
 +
#top-section {
 +
    width: 965px;
 +
    height: 0;
 +
    margin: 0 auto;
 +
    padding: 0;
 +
    border: none;}
 +
    body {
 +
    margin: 10px 0 0 0;
 +
    padding: 0;}
 +
#top-section {
 +
    width: 965px;
 +
    height: 0;
 +
    margin: 0 auto;
 +
    padding: 0;
 +
    border: none;}
 +
#menubar {
 +
        font-family: arial, sans-serif;
 +
        z-index: 9999;
 +
    font-size: 65%;
 +
    top: 5px;
 +
    height: 50px;}
 +
.left-menu:hover {
 +
    background-color: transparent;}
 +
#menubar li a {
 +
    background-color: transparent;}
 +
#menubar:hover {
 +
    color: white;}
 +
#menubar li a {
 +
    color: transparent;}
 +
#menubar:hover li a {
 +
    color: white;}
 +
    /* Removes "teams" from the menubar */
 +
#menubar > ul > li:last-child {
 +
    display: none;}
 +
/* Resizes the menubar to fik the links (default is 400px) */
 +
#menubar {
 +
    width: auto;}
 +
 
 +
    #contentSub, #search-controls, .firstHeading, #footer-box, #catlinks, #p-logo {
 +
    display:none;}
 +
#top-section {
 +
    border: none;
 +
    height: 0px;}
 +
#content {
 +
    border: none;
 +
    background-color: transparent;}
 +
 
 +
    #bodyContent {
 +
        background-color: transparent;
 +
    }
 +
 
 +
 
 +
 
  /*Page specific css*/
  /*Page specific css*/
   
   

Revision as of 08:43, 7 September 2013

Notebook //

  • human practices
    •
  • notebook
    •
    notebook
    • Week 1
    • Week 2
    • Week 3
    • Week 4
    • Week 5
    • Week 6
    • Week 7
    • Week 8
    • Week 9
    • Week 10
    • Week 11
    • Week 12
    • Week 13
    • Week 14
    • Week 15
    • Week 16
    • Week 17
    • Week 18

    WEEK 1

    June 4 2013 - June 11 2013

  • 4-Jun
    1. Learned how to make competent cells, growing up two strains for tomorrow
    2. Transformed 8 plasmids
    3. Determined EL222 fusion is risky but still going ahead with it
    4. Linkers are totally setlled
    5. Found zinc finger plasmid and updated target sequence
    6. Learned how to make tetr- mcherry fusion
    7. Settled on 5 promoters

  • 5-Jun
    1. Learned how to make competent cells, testing them and then making more tomorrow
    2. Transformed 8 plasmids again
    3. Made primers to clone the TET-GFP reporter system, the mCherry promoter strength system, ready to order
    4. Made ultramers for variable promoter blocks (and no target neg controls) – ready to order
    5. Spilled a lot of iced tea outside, bummer
    6. Started primers for dna binding machines
    7. Got a handle on cas9 fusions (pun intended).
    8. Put awesome pics in dropbox

  • 6-Jun
    1. Clean up dropbox
    2. Update budget sheet with addgene and cell center orders
    3. Finish primers for fusion
    4. Set up plate reader for GFP and mCherry assays
    5. run minipreps on pdawn, pdawn-mcherry, pet26b
    6. Grow up mCherry stock
    7. Wrote Penn iGEM on our plasmid

  • 7-Jun
    1. Transform
      1. C0012 –amp/chlor (do both)
      2. M11307 – amp/chlor (do both)
      3. I13458 – amp/chlor (do both)
      4. R0010 – amp/chlor (do both)
      5. R0051 – amp
      6. K206000 –chlor
    2. Start the LIMS and file all the strains and DNA we have made/ ordered

  • 8-Jun
    1. Miniprep Addgene stuff + transformations that worked
    2. Growing up low copy plasmids in 40mLs
  • Mini-prepped
    1. I9002
    2. I13458
    3. C0051
    4. Pdawn-mcherry
    5. Pdawn
    6. Dhsa mcherry
    7. Pdawn dhsa
    8. Psb1a3
    9. JM mcherry
    10. Pet26b

    WEEK 2

    June 11 2013 - June 18 2013

  • 17-Jun
    1. Grow up luxI culture and grow up tetR culture
    2. Sequence all of the minipreps
    3. Transform t9002 in psb1A3 in NEB10
    4. Retransform ptetGFP to see if BL21DE3 cells are competent
    5. Transform r0079, k081015, r0063 in NEB10
    6. Miniprep psb1k3
    7. Redo dam gel with more dna
    8. Figure out second control zfp from addgene
    9. Figure out how to add luxR binding site to target region
    10. Order sequencing primers for all addgene minipreps
    11. Bisulfite converted msssi methylated c0051

  • 18-Jun
    1. The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results
    2. Miniprep c0078, c0079 + make glycerol stocks check same plasmid with our kit
    3. Order 13420 (second zfp)
    4. 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)

  • 19-Jun
    1. Transform failed transformation
    2. Make competent DH5a && Dam-
    3. Figure out methylation assays for promoters
    4. Miniprep psb1A3 && all the 40mL cultures
    5. Picked many colonies
    6. Check pTet-gfp under blue light

    • WEEK 3

    June 18 2013 - June 25 2013

  • 21-Jun
    1. troubleshoot plux-luxI pcr
    2. roubleshoot pdawn-luxI pcr
    3. made pDawn-tetR pcr work
    4. troubleshoot pet26b-tetR pcr
    5. troubleshoot pDawn-GFP pcr
    6. troubleshoot pDawn-mCherry-secretion tag pcr
    7. miniprep growing cultures, be sure to pick only the glowing ligations
    8. ransform the correct t9002 amp ligation - determined from gel
    9. digested t9002 in amp and ptet gfp in amp to identify the correct ligation
    10. all of the chosen ptet gfp ligations worked, but let's not use 5 || 13 12. troubleshoot t9002 digest
    11. troubleshoot t9002 digest
      1. check for contamination of something (run uncut sample, sample + buffer, sample + 1 enzyme, sample + other enzyme, sample + both enzymes)

  • 24-Jun
    1. Dam-/dh5a v dam methylation + dpnI && dpnII digest
    2. Digested/ligated/transformed t9002 in amp
    3. Get methylated biobrick sequenced
    4. get chlor backbones sequenced
    5. culture t9002 transformations in liquid media with i751250
    6. mini prep stuff in the incubator
    7. figure out the primer issues 8
    8. Pick t9002 colonies for miniprep

    • WEEK 4

    June 25 2013 - July 2 2013

    • 26-Jun
      1. Dam-/dh5a v dam methylation + dpnI && dpnII digest
      2. Digested/ligated/transformed t9002 in amp
      3. get chlor backbones sequenced
      4. culture t9002 transformations in liquid media with i751250
      5. mini prep stuff in the incubator
      6. figure out the primer issues
      7. Pick t9002 colonies for miniprep
      8. USER Cloning reporter plasmid

    • 1-Jul
      1. Beautiful Brady Bunch photoshoot
      2. Troubleshooted and Re-tred PCR for user ends for reporter plasmid
      3. 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).
      4. Get methylated biobrick sequenced
      5. Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11
      6. Check if plux/luxI system is working in liquid cultures – this failed
        1. a. Might be strain competition, need to know growth rates
      7. Re-suspend primers for lux amplifier
      8. Mini-prep: e0040, psb1a3, r0062

    • 2-Jul

      1. Think about application of mathylation project in e.coli
      2. Ceck if plux/GFP-psb1C3 system is working in liquid cultures
        1. +/- AHL induction at 100nM
        2. Compare with ptetGFP fluorescence, normal LB fluorescence
      3. Streak zinc finger 2
      4. Grow up 44251
      5. Transform up R0062
      6. When BstuI arrives
        1. Assay BstuI working
        2. Assay the bvluc and tale1 minipreps in dh5a, dam-, and bl21 cells with msssi and bstuI
        3. Results: BstUI is blocked by methylation, and cells don’t normally methylate
      7. Growing up t9002 in chlor and i751250 in amp for fluorescence study
      8. Investigate CHIP or other ways of determining DNA binding domain specificity

    WEEK 5

    July 2 2013 - July 9 2013

    • 3-Jul
      1. Streak zinc finger 2
      2. Grow up 44251
      3. Look into lux box being light sensitive
    • 4-Jul
      1. Mini prep 44251
      2. Pick ZFP2 colonies to grow up, throw out liquid culture in fridge
    • 5-Jul
      1. Repeat BstUI assay, taking into account new controls
      2. Suspend the primers in the freezer
      3. We need to check if the origin of replications are compatible before co transformation
      4. Characterize pDawn-mcherry
      5. Practice measuring fluorescence
    • 6-Jul
      1. troubleshoot ptetGFP user PCR - band was visible but too small to extract
      2. gel extract promoter fragments from USER PCR
      3. re-do USER PCR for: TetR, pTetGFP
      4. Design/check/order (Tale,Cas,ZFP)-flex-REV primers - check fwd primers

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