Team:UPenn/Notebook

From 2013.igem.org

(Difference between revisions)
Line 1: Line 1:
-
<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01//EN"
+
<html>
-
    "http://www.w3.org/TR/html4/strict.dtd"
+
-
    >
+
-
<html lang="en">
+
<head>
<head>
 +
<script src="https://googledrive.com/host/0B4ZBZOYYKBzETkFqdnhMeV9fMzA" ></script>
 +
     <style>
     <style>
 +
 +
.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;
 +
}
 +
 +
#menubar {
 +
    display: none;
 +
    position: absolute;
 +
}
 +
.noprint {
 +
    display: none;
 +
    position: absolute;
 +
}
 +
#footer {
 +
    display: none;
 +
    position: absolute;
 +
}
 +
 +
/*sections of page*/
 +
 +
/*#banner {
 +
width: 100%;
 +
background-color: #B4EEFF;
 +
height: 300px;
 +
}*/
 +
 +
.space {
 +
height: 20px;
 +
background-color: gray;
 +
}
 +
 +
.one {
 +
height:600px;
 +
position: relative;
 +
padding-top: 30px;
 +
width: 100%;
 +
background-color: black;
 +
background-image: url("https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/Yn0SK0Y0-7/snap.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg");
 +
background-position: 300px 50px;
 +
background-repeat: no-repeat;
 +
background-size: 90%;
 +
}
 +
 +
.circle {
 +
    height: 20px;
 +
    width: 20px;
 +
    background-color: black;
 +
    -moz-border-radius: 100%;
 +
    -ms-border-radius: 100%;
 +
    -webkit-border-radius: 100%;
 +
    -o-border-radius: 100%;
 +
    border-radius: 100%;
 +
}
 +
 +
.two {
 +
height:600px;
 +
position: relative;
 +
padding-top: 30px;
 +
width: 100%;
 +
background-color: #50393F;
 +
background-image: url("https://dl.dropboxusercontent.com/sh/h4cxid18rzzjgan/-ZtRJ7Gu1U/photo-8.JPG?token_hash=AAGlfzG2xZpKrOTI8pyH02EbYxY1vK3QLUWKsWuYdaPbYg");
 +
background-position: 300px -100px;
 +
background-repeat: no-repeat;
 +
background-size: 80%;
 +
}
 +
 +
.three {
 +
position: relative;
 +
height:1000px;
 +
width: 100%;
 +
background-color: rgb(200,200,200);
 +
}
 +
 +
.right {
 +
    float:right;
 +
    text-align: left;
 +
    margin-right: 200px;
 +
    margin-top: -300px;
 +
    background-color: white;
 +
    height: 200px;
 +
    padding:20px;
 +
    width: 30%;
 +
    -ms-filter: "progid:DXImageTransform.Microsoft.Alpha(Opacity=70)";
 +
  filter: alpha(opacity=70);
 +
-moz-opacity: 0.7;
 +
-khtml-opacity: 0.7;
 +
  opacity: 0.7;
 +
}
 +
 +
.wrapper {
 +
  background-color: rgb(230,230,230);
 +
  opacity: 1;
 +
  height: 500px;
 +
  width: 90%;
 +
  float: center;
 +
  margin: 50px;
 +
}
 +
 +
         #accordion-container {
         #accordion-container {
font-size: 13px;
font-size: 13px;
Line 70: Line 197:
     </style>
     </style>
<script type="text/javascript">
<script type="text/javascript">
-
   
 
-
   
 
-
   
 
-
   
 
-
   
 
-
   
 
-
   
 
-
   
 
      
      
      
      
Line 253: Line 372:
<li>Finalized synthesis order (minus linker)</li>
<li>Finalized synthesis order (minus linker)</li>
</ul>
</ul>
-
 
-
 
-
</div>
 
-
<h2 class="accordion-header">Week 4</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
 
-
<p><b>June 22</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab</p>
 
-
<ul>
 
-
<li>Ashwin - Repeated miniprep on pDawn and did test cut</li>
 
-
<li>Peter  - Miniprepped pet26b and digested with BglII and EcorRI</li>
 
-
<li>Avin - Miniprepped pet26b, made glycerol stock and digested with BamH1 and Not1</li>
 
-
</ul>
 
-
&nbsp;&nbsp;<p>Dry Lab</p>
 
-
<ul>
 
-
<li>Avin - Finalized and sent in synthesis order (still awaiting order confirmation)</li>
 
-
</ul>
 
-
 
-
<p><b>June 25</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab</p>
 
-
<ul>
 
-
<li>Ashwin/Avin - Column purification, ligation, and transformation of Pet26b-mCherry</li>
 
-
<li>Peter -run gel of eGFP/plsr ligation</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab</p>
 
-
<ul>
 
-
<li>Avin - Sent in final gene synthesis order</li>
 
-
<li>Mike - reviewed pDawn protocol, reviewed TetR sequences</li>
 
-
<li>Peter- Order restriction enzymes from cell center</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>June 26</b></p>
 
-
&nbsp;&nbsp;<p>Wet Lab</p>
 
-
<ul>
 
-
<li>Avin - Picked colonies for Pet26b-mCherry and pJT106b</li>
 
-
<li>Mike/Ashwin - Plated pJT122</li>
 
-
</ul>
 
-
&nbsp;&nbsp;<p>Dry Lab</p>
 
-
<ul>
 
-
<li>Everyone - Brainstormed human practices, Wiki design</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
<p><b>June 27</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Miniprepped pet26b-mCherry and pDawn-mCherry</li>
 
-
<li>Test cut pet26b-mCherry with HindIII and ClaI (picture temporarily saved in MEAM folder of pqiao account), bands looked okay, chose C2 for remainder of experiments and made a glycerol stock, transformed both pet26b-mCherry and pDawn-mCherry into BL21(DE3)- Gold (10 ul cells, ~1ng DNA)
 
-
Plated 200 ul and 20 ul</li>
 
-
<li>Inoculated colonies for pJT122, pJT106b, PhyB (BBa), PIF3 (BBa), and Cph8 (BBa)</li>
 
-
</ul>
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Started exploring possible wiki designs and coding</li>
 
-
</ul>
 
-
<br>
 
-
<p><b>June 28</b></p>
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Miniprepped pJT122, pJT106b, PhyB, PIF3, Cph8, made glycerol stocks</li>
 
-
<li>Picked colonies for pET26b-mCherry and pDawn-mCherry, innoculated, grew up, then diluted
 
-
Measured OD along the way</li>
 
-
</ul>
 
-
&nbsp; &nbsp; <p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Set up light and dark incubators</li>
 
-
</ul>
 
-
 
-
<p><b>June 29</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Tested pDawn-mCherry and pET-mCherry, pDawn-mCherry expressed mCherry only after light induction</li>
 
-
<li>Nanodropped pJT122, pJT106b, PhyB, PIF3, Cph8</li>
 
-
<li>Performed digest of pET-26b, eGFP, and plsr</li>
 
-
<li>Spread Biobrick shipment on Amp plates (lsrR, lsrK)</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Opened Bio-Rad shipments</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
</div>
 
-
</div>
 
-
<p style="text-align:center; color:white;">July 2012 Notebook</p>
 
-
<div id="accordion-container">
 
-
<h2 class="accordion-header">Week 5</h2>
 
-
 
-
<div class="accordion-content">
 
-
             
 
-
<p><b>July 2</b></p>
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Transformed ho1 and pcyA BioBricks</li>
 
-
<li>Peter: ligations</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
 
-
<li>Contacted labs for JT2 and pPL-PCB</li>
 
-
<li>Worked on Human Practices</li>
 
-
</ul>
 
-
 
-
<br>
 
-
 
-
 
-
 
-
<p><b>July 3</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab</p>
 
-
<ul>
 
-
<li>Nothing to be done for drug delivery</li>
 
-
</ul>
 
-
&nbsp;&nbsp;<p>Dry Lab</p>
 
-
<ul>
 
-
<li>Avin - digital schematic, helped with primers, read human practices bacterial therapy stuff</li>
 
-
<li>Mike - talked to Dan, developed cloning strategy for getting Cph8 into the pDawn backbone, started primer design</li>
 
-
</ul>
 
-
</br>
 
-
 
-
 
-
 
-
<p><b>July 5</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab</p>
 
-
<ul>
 
-
<li>Picked colonies of ho1 and pcyA</li>
 
-
<li>Inoculated pDawn-mCherry cells for timecourse</li>
 
-
</ul>
 
-
&nbsp;&nbsp;<p>Dry Lab</p>
 
-
<ul>
 
-
<li>worked on primer design/cloning strategy and talked to Dan</li>
 
-
<li>worked on schematic and wiki</li>
 
-
<li>worked on human practices</li>
 
-
</ul>
 
-
</br>
 
-
 
-
<p><b>July 6-7</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
 
-
<li>Took pDawn-mCherry time course</li>
 
-
<li>Sterilized incubator and TC hood</li>
 
-
<li>Moved lab stuff to small room</li>
 
-
<li>Redesigned primers for plsr &amp; egfp</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>
 
-
Contacted FDA contacts for human practices
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
</div>
 
-
 
-
<h2 class="accordion-header">Week 6</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
<p><b>July 9</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Inoculated 50mL cultures of pDawn-mCherry</li>
 
-
<li>Set up pH meter, picked up JT2</li>
 
-
<li>Organized lab area </li>
 
-
<li>Made bacterial streaks for biobricks</li>
 
-
</ul>
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Ordered mammalian cell culture media</li>
 
-
<li>Finalized wiki template</li>
 
-
<li>Worked on cloning steps for cph8</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
<p><b>July 10</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Did time course for pDawn-mCherry, but since it was from a glycerol stock, saw no growth ? will let grow overnight, then dilute in the morning and start a new time course</li>
 
-
<li>Picked colonies from transformed Biobricks and innoculated</li>
 
-
<li>Resuspended ClyA and LuxS IDT DNA and transformed into DH5alpha</li>
 
-
<li>TC/Sterile practice training</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Ordered primers for ClyA-RFP, Peters primers</li>
 
-
<li>Worked on wiki</li>
 
-
<li>Emailed Tabor for pJT106b plasmid map</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
<p><b>July 11</b></p>
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>
 
-
Recorded BL21 pDawn-mCherry growth curve and calculated doubling time</li>
 
-
<li>
 
-
<ul> Miniprepped biobricks
 
-
<li>
 
-
BBa_K265008 - Ice Nucleation Protein NC
 
-
</li>
 
-
<li>
 
-
BBa_K523013 - Plac + INP-EYFP
 
-
</li>
 
-
<li>
 
-
BBa_K299810 - B0032 + Invasin
 
-
</li>
 
-
<li>
 
-
BBa_K257010 - ClyA + RFP
 
-
</li>
 
-
</ul>
 
-
</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Worked on biofilm project schematic</li>
 
-
<li>More work on wiki</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
<p><b>July 13</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Diluted to OD600=0.01 and take 0-8hr time points of pDawn-mCherry fluorescence time course</li>
 
-
<li>Thawed out HEK293T cells</li>
 
-
</ul>
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Worked on wiki/schematic</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>July 17</b><p>
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
 
-
<li>Re-ligation of pDawn with IDTClyA and ClyA-RFP, Transformation into DH5alpha/XO1blue</li>
 
-
<li>Performed PCR of plsr &amp; gfp w/new primers.</li>
 
-
<li>Picked colonies of T7 and INPNC-DARPin</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Designed experiments for ClyA and INPNC-DARPin assays</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
</div>
 
-
 
-
<h2 class="accordion-header">Week 7</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
<p><b>July 16</b></p>
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>
 
-
Transformed INPNC-DARPin and T7 BioBrick
 
-
</li>
 
-
</ul>
 
-
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>SAAST presentation</li>
 
-
<li>Met with Lazzara lab to discuss DARPin experiments</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>July 18</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>
 
-
Re-inoculated T7 and INPNC-DARPin</li>
 
-
<li>PCR ClyA-RFP, Gel purify PCR ClyA-RFP product</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Worked on DARPin assay experiment design</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>July 19</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Trypan Blue Assay</li>
 
-
<li>Digested pDawn-mCherry with BamHI, NotI, pDawn-mCherry with BamHI,Digest pDawn-mCherry with NotI, Digested IDTsmart-ClyA with BamHI, NotI (Gel for Digests showed possible degeneration of NotI enzyme...) </li>
 
-
<li>Ligated pDawn backbone (old) with 1) ClyA and 2) ClyA-RFP and Ligated pDawn backbone (new) with ClyA and ClyA-RFP, as well as pDawn old backbone with H20 and pDawn new backbone with H20 (ligation controls)</li>
 
-
<li>Transformed all of above ligations into DH5alpha</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
<p><b>July 23</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Ashwin-Design test cuts for pDawn-Clya, pDawn-ClyA+RFP </li>
 
-
<li>Ashwin-Digest pDawn-ClyA+pDawn-ClyA+RFP, Run on gel </li>
 
-
<li>Nikita - Miniprep pDawn culture in Sarkar cold room</li>
 
-
<li>Digest of pET-26b w/ BglII, EcoRI-Peter</li>
 
-
<li>Digest of plsr product w/ BglII, PseI-Peter</li>
 
-
<li>Digest egfp w/ SpeI, EcoRI-Peter</li>
 
-
<li>Ligate &amp; transform pET-26-plsr-GFP-Peter</li>
 
-
<li>Digest INPNC-DARPin</li>
 
-
<li>Avin - Try to rescue 293T cells after CO2 arrives if can’t be rescued, thaw out more 293T</li>
 
-
<li>Transform pET-26b-luxS - Peter</li>
 
-
<li>Avin - pick GAVPO </li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Ordered AI-2 ASAP-peter</li>
 
-
<li>Nikita - Researched whether ClyA is toxic to E. coli BL21, JT2, DH5 alpha, and whether it is secreted in these strains</li>
 
-
<li>Read Daphne+Dr. Sarkar’s ligation paper </li>
 
-
<li><ul>Avin - Buy:
 
-
<li>DH5 alpha</li>
 
-
<li>20 kan plates, 10 amp plates</li>
 
-
<li>Cell Counter</li>
 
-
</ul>
 
-
</li>
 
-
<li>
 
-
Write out competent cell production protocol-Peter</li>
 
-
<li>Obtained BL21 transformation protocol from daphne/find it-Peter</li>
 
-
<li>Avin - Logo design, human practices</li>
 
-
<li>Ashwin - wiki</li>
 
-
</ul>
 
-
</br>
 
-
 
-
 
-
 
-
<p><b>July 24</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Pick colonies of pDawn-ClyA and pDawn-ClyA+RFP (DH5alpha)</li>
 
-
<li>Avin - Picked 2 colonies (light+dark) of pDawn-ClyA+RFP (BL21), set up pDawn culture experiment, check on HEK293T and passage into 6 well plates if confluent</li>
 
-
<li>Avin - Ran Gel, no DNA</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Peter: order the promoter (or figure out an alternative method..)</li>
 
-
<li>Ashwin: wiki</li>
 
-
<li>Avin - Edit/Order primers for HA tag and His tag, design pDawn-ClyA experiments, human practices</li>
 
-
<li>Nikita - Write human practices</li>
 
-
</ul>
 
-
</br>
 
-
 
-
 
-
</div>
 
-
<h2 class="accordion-header">Week 8</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
<p><b>July 26</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Avin - Finish ClyA-RFP time course, collect ClyA supernatant, measure fluorescence of ClyA-RFP triplicates, spin down + pictures if red, Miniprep of DARPin, possible ClyA assay</li>
 
-
<li>Peter- ligation, transformation, plating of ligations, help Avin with ClyA cell lysis experiments </li>
 
-
<li>Transform luxS6-8 into BL21 </li>
 
-
<li> Nikita - Miniprep GAVPO and DARPin </li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Avin - Call IDT at 9am, order blood agar plates, nissl stuff after we do background research</li>
 
-
<li>Ashwin - wiki, research getting recombinant ClyA</li>
 
-
<li>Nikita - Human practices, Nissle research, plan experiments</li>
 
-
<li><ul>Peter
 
-
<li>Complete FDA writeup</li>
 
-
<li>Order ATCC Strains</li>
 
-
<li>Order lss primers</li>
 
-
<li>Order AI-2</li>
 
-
<li>Order Crystal Violet Stain</li>
 
-
<li>Plan ClyA experiment in JMOutline</li>
 
-
</ul>
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
<p><b>July 30</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>Avin - Transform pBAD33 and pSB4A5, Inoculate BL21 Intein colonies</li>
 
-
<li>Redo the test cuts for pDawn and pDawn-ClyA-RFP with XmaI and ClaI on a 0.7% gel</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Avin - Human Practices</li>
 
-
<li><ul>Mike - Primers
 
-
<li>pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, figure out reverse</li>
 
-
<li>pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, figure out reverse</li>
 
-
<li>pet26b-ClyA+RFP keeping pelB with 6xHis C terminus - use NcoI on forward primer, figure out reverse</li>
 
-
<li>pet26B-CLlyA+RFP removing pelB with 6x His C terminus - use NdeI on forward primer, figure out reverse</li>
 
-
<li>fix frame shift in pDawn and include 6xHis N terminus - use NdeI on forward primer, figure out reverse</li>
 
-
<li>ClyA </li>
 
-
<li>mCherry</li>
 
-
<li>ClyA-RFP</li>
 
-
<li>Cph8 primers - figure out pJT106b primers</li>
 
-
</ul>
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>July 31</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>
 
-
Test Cut pDawn-ClyA-RFP colony 4 w/ XmaI and Cla</li>
 
-
<li>
 
-
Transform the pDawn-ClyA-RFP colony 4 plasmid (assuming test cuts look good) into BL21 and Dh5alpha</li>
 
-
<li>
 
-
Check for growth of Intein-mCherry plasmid</li>
 
-
<li><ul>
 
-
Assuming growth:
 
-
<li>miniprep DH5alpha culture</li>
 
-
<li>induce BL21 culture with IPTG - see Jordan for protocol</li>
 
-
dilute in morning, when it gets to 0.8, induce with IPTG (what concentration) and then in a few hours (2-4), can spin down and see pellet</li>
 
-
<li>If primers arrive ? PCR (x2) for DARPin (check order status for IDT in the morning, call cell center if it says its been shipped)</li>
 
-
<li>Pick colonies and innoculate pBAD33 (chloramphenicol) and pSB4A5 (ampicillin)</li>
 
-
</ul>
 
-
</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Mike - Order Primers<ul></li>
 
-
<li>
 
-
pET26b-ClyA (IDT) keeping pelB with 6xHis C terminus - use NcoI on forward primer, XhoI on reverse (no stop codon)</li>
 
-
<li>
 
-
pET26b-ClyA (IDT) removing pelB with 6xHis C terminus - use NdeI on forward primer, XhoI on reverse (no stop codon)
 
-
</li>
 
-
<li>
 
-
fix frame shift in pET26b for ClyA-RFP (NdeI on forward, NotI on reverse, include stop) - can’t use NdeI because there is an NdeI cut site inside ClyA-RFP
 
-
</li>
 
-
<li>
 
-
fix frame shift in pDawn and include 6xHis N terminus
 
-
</li>
 
-
<li>
 
-
ClyA - NdeI on forward, BamHI on reverse
 
-
</li>
 
-
<li>
 
-
mCherry - NdeI on forward, NotI on reverse
 
-
</li>
 
-
<li>
 
-
ClyA-RFP - NdeI on forward, NotI on reverse - can’t use NdeI because there’s an NdeI cut site inside ClyA-RFP
 
-
</li>
 
-
<li>
 
-
Cph8 primers - figure out pJT106b primers
 
-
</li>
 
-
</ul>
 
-
</li>
 
-
<li>
 
-
Mike - Take out Biohazard for Sevile
 
-
</li>
 
-
<li>
 
-
Ashwin - wiki
 
-
</li>
 
-
</ul>
 
-
 
-
<br>
 
-
 
-
 
-
</div>
 
-
</div>
 
-
 
-
<p style="text-align:center;color:white;">August 2012 Notebook</p>
 
-
<div id="accordion-container">
 
-
<h2 class="accordion-header">Week 9</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
<p><b>August 1st</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<li>pET-26-plsr-gfp triple ligation ( (+) indicates positive control, (-) indicates negative control)</li>
 
-
<li><ul>
 
-
Gel Purification 8:30-12:00
 
-
<li>
 
-
1% gel 50mL +5uL SyberSafe</li>
 
-
<li>
 
-
pET-26b cut w/ EcoRI-HF (+)</li>
 
-
<li>
 
-
pET-26b cut w/ EcoRI-HF &amp; BglII</li>
 
-
<li>
 
-
run against pET-26b uncut for reference
 
-
</li></ul></li>
 
-
<li><ul>
 
-
Ligation (Start w/ 25ng vector) 12:00-3:00
 
-
<li>
 
-
Vector:GFP:plsr</li>
 
-
<li>1:6:6</li>
 
-
<li>1:4:4</li>
 
-
<li>1:1:1</li>
 
-
<li>pET-26b cut w/ EcoRI-HF (+)</li>
 
-
<li>pET-26b cut w/ EcoRI-HF &amp; BglII (-)</li>
 
-
<li>INCUBATE @ RT 1hr</li>
 
-
<li><ul>
 
-
Transform Into DH5a Max Efficiency 3:00-6:00
 
-
<li>Transform pET-26b (+)</li>
 
-
<li>Transform H2O (-)</li>
 
-
<li>Transform 1:6:6</li>
 
-
<li>Transform 1:4:4</li>
 
-
<li>Transform 1:1:1</li>
 
-
</ul></li>
 
-
<li>
 
-
Total Plates Needed=8</li>
 
-
<li>
 
-
Check IDT primer order in the morning</li>
 
-
<li>
 
-
Avin - Pick colonies of pDawn-ClyA-RFP (DH5a and BL21) and inoculate into 5 mL LB culture
 
-
</li>
 
-
<li>
 
-
Avin - Miniprep pSB4A5, pBAD33, Intein-mCherry (DH5a)
 
-
Grow up BL21 Intein-mCherry and induce for fun?
 
-
</li>
 
-
</ul>
 
-
</br>
 
-
 
-
<p><b>August 2</b></p>
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>
 
-
pDawn-ClyA-RFP Time course - Avin </li>
 
-
<li>
 
-
growth to 0.8 and 1:1000 IPTG induction of 50mL BL21 Intein-mCherry - Avin </li>
 
-
<li>
 
-
Miniprep of DH5 alpha pDawn-ClyA-RFP - Ashwin </li>
 
-
<li>
 
-
PCR purify INPNC-DARPin-HA Tag - Mike </li>
 
-
<li>
 
-
Digest INPNC-DARPin-HA Tag and pET26b with EcoRI and NdeI
 
-
</li>
 
-
<li>
 
-
Column purify INPNC-DARPin-HA Tag</li>
 
-
<li>
 
-
Gel purify pET26b </li>
 
-
<li>
 
-
Digest INPNC, INPNC-HA Tag, 6xHis-DARPin-Ha Tag with NdeI and BamHI </li>
 
-
<li>Column purify INPNC, INPNC-HA Tag, 6xHis-DARPin-HA Tag</li>
 
-
<li>
 
-
Gel purify PET26b</li>
 
-
<li>
 
-
Gel purification of pET-26b digested with BglII and EcoRI-HF </li>
 
-
<li>
 
-
Plated S. Epidermis and E. Coli containing Lysostaphin</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Logo - Avin</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>August 3</b></p>
 
-
 
-
&nbsp;&nbsp;<p>Wet Lab:</p>
 
-
<ul>
 
-
<li>BL21 pDawn-ClyA-RFP time course, store blood agar plates in fridge - Avin</li>
 
-
<li>BL21 Intein-mCherry Protein purification and Coomassie gel - Avin/Peter</li>
 
-
<li>Ligate and transform pET26b-INPNC, pET26b-INPNC-HATag, pET26b-INPNC-DARPin-HATag, pET26b-6xHis-DARPin-HATag</li>
 
-
<li>Saturday - pick colonies</li>
 
-
<li>Sunday/Monday - Miniprep, test cuts, transformation</li>
 
-
<li>Check if ClyA primers are shipped, if so pick up at cell center</li>
 
-
<li>PCR ClyA with various primers</li>
 
-
<li>Run on Gel, Gel Purify, Digest, Column Purify, Ligate, Transform</li>
 
-
</ul>
 
-
&nbsp;&nbsp;<p>Dry Lab:</p>
 
-
<ul>
 
-
<li>Pick up primers</li>
 
-
<li>Meeting with Dr. Sarkar</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
 
-
                        </div>
 
-
 
-
<h2 class="accordion-header">Week 10</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
<p><b>August 13</b></p>
 
-
<ul>
 
-
<li>
 
-
Started pDawn ClyA &amp; pDawn ClyA-RFP cultures for blood agar experiment</li>
 
-
<li>
 
-
Meeting with Dr. Sarkar to discuss progress on both projects</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
<p><b>August 14 </b></p>
 
-
<ul>
 
-
<li><ul>
 
-
Reviewed sequencing results and transformed the following into BL21 and DH5 alpha:
 
-
<li>
 
-
pET26b-INPNC
 
-
</li>
 
-
<li>
 
-
pET26b-INPNC-HA
 
-
</li>
 
-
<li>
 
-
pET26b-6xHis-DARPin-HA
 
-
</li>
 
-
<li>
 
-
pET26b-INPNC-DARPin-HA
 
-
</li>
 
-
<li>
 
-
pET26b-ClyA-6xHis
 
-
</li>
 
-
<li>
 
-
pET26b-PelB-ClyA-6xHis
 
-
</li>
 
-
<li>
 
-
pDawn-ClyA-6xHis
 
-
</li></ul></li>
 
-
<li>
 
-
Serial dilution and plating of pDawn ClyA &amp; pDawn ClyA-RFP cultures for blood agar experiment</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
 
-
 
-
                        </div>
 
-
 
-
<h2 class="accordion-header">Week 11</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
 
-
<p><b>August 15</b></p>
 
-
<ul>
 
-
<li>Re-plated all constructs</li>
 
-
<li>pDawn-ClyA and pDawn-ClyA-RFP demonstrated light-dependent hemolysis!</li>
 
-
<li>BL21 washing titration experiment for flow cytometry experiment - obtained optimal starting OD600 of 0.05 to obtain ~1E6 cells after all washes and incubations</li>
 
-
<li>Made Incubation Buffer for flow cytometry experiment</li>
 
-
<li>Designed/ordered pBAD33-eGFP Primers</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
<p><b>August 16</b></p>
 
-
<ul>
 
-
<li>
 
-
Picked colonies on all BL21 and DH5 constructs, including inducing and non-inducing conditions, set up flow cytometry stuff, booked flow core
 
-
</li>
 
-
<li>
 
-
Started cultures of pDawn-ClyA, pDawn-ClyA-RFP, and pDawn-ClyA-mCherry for repeat blood agar experiment</li>
 
-
<li>Set up light incubator properly</li>
 
-
<li>Started protein purification cultures of pDawn-ClyA-6xHis, pET26b-ClyA-6xHis, and pET26b-PelB-ClyA-6xHis, grew to OD600=0.8, induced with IPTG or light</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
<p><b>August 22 </b></p>
 
-
<ul>
 
-
<li>
 
-
Image INPNC-DARPin-HA, INPNC-HA, DARPin-HA (both induced and not induced) on the confocal - Avin
 
-
</li>
 
-
<li>
 
-
Figure out ClyA imaging system and maybe design a stencil (not a high priority)
 
-
</li>
 
-
<li>
 
-
Pick up media and everything else from cell center - Mike
 
-
</li>
 
-
<li>
 
-
Ligate ClyA into lactococcus? -- Talk to Daphne about vectors
 
-
</li>
 
-
<li>
 
-
Order the cytotoxicity assay from Promega - Avin
 
-
</li>
 
-
<li>
 
-
Ask Dr. Sarkar for cell center account - Mike
 
-
</li>
 
-
<li>
 
-
Order pDawn sequencing primer - Avin
 
-
</li>
 
-
<li>
 
-
Run GFP PCR product on Gel,
 
-
Gel didn’t work ? rerun PCR using gradient annealing temp
 
-
Run PCR products on gel, check if works
 
-
</li>
 
-
<li>
 
-
Look into biobrick format - Mike
 
-
</li>
 
-
<li>
 
-
Make an in-depth plan for the incoming weeks, prioritizing what needs to be done
 
-
Cloning! what is the final construct? Design and execute
 
-
add RBS to pBAD33 primer
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>Plan for August 23</b></p>
 
-
<ul>
 
-
<li>
 
-
Add RBS/SD to pBAD33 forward primer and re-order it
 
-
</li>
 
-
<li>
 
-
Send out sequencing for INPNC-DARPin-HA (and what else?)
 
-
</li>
 
-
<li>
 
-
Look at old sequencing data just to make sure everything worked
 
-
</li>
 
-
<li>
 
-
Pick up IPTG from the cell center and make stock solution/aliquots
 
-
</li>
 
-
<li>
 
-
Using new IPTG, spread on blood agar plates (final dilution of 1 to 1000)
 
-
</li>
 
-
<li>
 
-
Spread Kan on Blood Agar Plates
 
-
</li>
 
-
<li>
 
-
Plate pET26b-ClyA-His (+/-), pET26b-pelB-ClyA-His (+/-), and pDawn-ClyA(FS) (+/-) on Blood Agar + Kan plates
 
-
</li>
 
-
<li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
<p><b>August 24</b></p>
 
-
<ul>
 
-
<li>
 
-
If colonies were picked, miniprep, then test cut, then run on diagnostic gel ? transform into BL21</li>
 
-
<li>
 
-
If colonies were not picked, then pick colonies</li>
 
-
<li>
 
-
Analyze sequencing results</li>
 
-
<li>
 
-
Plan out INPNC-DARPin-HA experiments
 
-
</li>
 
-
<li>
 
-
Figure out cloning for 1 plasmid system
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
 
-
  </div>
 
-
 
-
<h2 class="accordion-header">Week 12</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
<p><b>Goals in order of priority:</b></p>
 
-
<ul>
 
-
<li>
 
-
Show that INPNC-DARPin-HA binds to HER2 in vitro
 
-
</li>
 
-
<li>
 
-
Show that purified 6xHis-DARPin-HA binds to Her2 - Monday?
 
-
</li>
 
-
<li>
 
-
Show that INPNC-DARPin-HA is displayed on the surface
 
-
</li>
 
-
<li>
 
-
Show INPNC-mCherry is displayed on surface (confocal?)
 
-
</li>
 
-
<li>
 
-
Construct ClyA and ClyA-His BioBricks
 
-
</li>
 
-
<li>
 
-
Construct INPNC-mCherry Biobrick
 
-
</li>
 
-
<li>
 
-
Construct Surface Display BioBrick (need INPNC-mCherry to work)
 
-
</li>
 
-
<li>
 
-
Transform ClyA into Lactococcus and plate onto blood agar for Human Practices-
 
-
</li>
 
-
<li>
 
-
Stencil experiment showing spatial control of pDawn-ClyA (could be done next week, only takes a little time and
 
-
artistic ability)
 
-
</li>
 
-
<li>
 
-
Show that cph8 works with a reporter
 
-
</li>
 
-
<li>
 
-
Show that cph8-ClyA works
 
-
</li>
 
-
</ul>
 
-
 
-
<p><b>Experiments in order of priority:</b></p>
 
-
<ul>
 
-
<li>
 
-
Construction of pBAD33-eGFP (Mike)
 
-
</li>
 
-
<li>
 
-
Order primers for ClyA-His and ClyA to clone them into BioBrick backbone
 
-
</li>
 
-
<li>
 
-
Order primers for INPNC-mCherry
 
-
</li>
 
-
<li>
 
-
Order primers for cph8-reporter, cph8-clyA-his
 
-
</li>
 
-
<li>
 
-
SKBR3 imaging with pBAD33-eGFP/pET26b-INPNC-DARPin-HA bacteria on confocal
 
-
</li>
 
-
<li>
 
-
INPNC-DARPin immunos, 0.2mM IPTG, overnight induction
 
-
</li>
 
-
<li>
 
-
INPNC-DARPin bacterial flow cytometry on FACS machine (can use same bacteria as immuno)
 
-
</li>
 
-
<li>
 
-
Create stable cell line of SKBR3-mCherry (Jordan)
 
-
</li>
 
-
<li>
 
-
Obtain Lactococcus expression vector, order primers to clone in ClyA-His
 
-
</li>
 
-
<li>
 
-
Design surface display biobrick
 
-
</li>
 
-
<li>
 
-
Cytotoxicity experiment on SKBR3</li>
 
-
<li>
 
-
Protein purification on His-DARPin-HA (Peter)
 
-
</li>
 
-
<li>
 
-
Flow cytometry of SKBR3 cells incubated with his-DARPin-HA (someone/Najaf)
 
-
</li>
 
-
<li>
 
-
Construct cph8-ClyA plasmid
 
-
</li>
 
-
<li>
 
-
cph8-reporter timecourse
 
-
</li>
 
-
<li>
 
-
cph8-ClyA blood agar
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>August 27</b></p>
 
-
<ul>
 
-
<li>
 
-
PCR eGFP out of PHAT (annealing = 65), run on 1% gel, gel purify
 
-
</li>
 
-
<li>
 
-
Digest eGFP and pBAD33 with PstI and XmaI, column purify
 
-
</li>
 
-
<li>
 
-
Ligate at RT for 1 hour, transform into DH5a
 
-
depending on time, otherwise ligate at 16C/4C overnight and transform the next day
 
-
</li>
 
-
<li>
 
-
Order Primers for (in order of priority)
 
-
</li>
 
-
<li>
 
-
ClyA and ClyA-His Biobricks
 
-
</li>
 
-
<li>
 
-
INPNC-mCherry construct
 
-
</li>
 
-
<li>
 
-
ClyA into lactococcus plasmid
 
-
</li>
 
-
<li>
 
-
Cph8-reporter plasmid
 
-
</li>
 
-
<li>
 
-
Cph8-ClyA-His plasmid
 
-
</li>
 
-
<li>
 
-
INPNC surface display vector (provided it works)
 
-
</li>
 
-
<li>
 
-
Start cell culture of SK-BR-3 cells?
 
-
</li>
 
-
<li>
 
-
Get protocols
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>August 28</b></p>
 
-
<ul>
 
-
<li>
 
-
start cultures for purification of His-DARPin-HA
 
-
</li>
 
-
<li>
 
-
HA affinity purification of INPNC-DARPin-HA?
 
-
</li>
 
-
<li>
 
-
Print and hand in safety form to EHRS
 
-
</li>
 
-
<li>
 
-
Pick up SK-BR-3 plate from Cal, learn protocols
 
-
</li>
 
-
<li>
 
-
Add FBS to our McCoy’s media
 
-
</li>
 
-
<li>
 
-
Design and order primers to put ClyA into lactococcus expression vector
 
-
</li>
 
-
<li>
 
-
Set up preliminary stencil experiment, send avin plate base diameter (approximate 86mm)
 
-
</li>
 
-
<li>
 
-
Design INPNC-mCherry-DARPin primers
 
-
</li>
 
-
<li>
 
-
pBAD33-eGFP
 
-
</li>
 
-
<li>
 
-
Run Gradient PCR  products (which ran overnight) on gel, gel purify the best band
 
-
</li>
 
-
<li>
 
-
Digest eGFP and pBAD33 with PstI and XmaI, column purify/gel purify
 
-
</li>
 
-
<li>
 
-
Ligate at RT for 1 hour, transform into DH5a
 
-
depending on time, otherwise ligate at 16C/4C overnight and transform the next day
 
-
</li>
 
-
<li>
 
-
Start DH5 alpha cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA</li><li> Also start cultures of pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>August 29</b></p>
 
-
<ul>
 
-
<li>
 
-
Induce His-DARPin-HA culture (100 microliters of 1 M IPTG)</li>
 
-
<li>
 
-
Monitor SK-BR-3 </li>
 
-
<li>
 
-
Primers for ClyA into lactococcus</li>
 
-
<li>
 
-
Primers for INPNC-mCherry-DARPin </li>
 
-
<li>
 
-
Pick colonies for pBAD33-eGFP if the RT ligation and transformation worked - Peter/Ashwin</li>
 
-
<li>
 
-
If RT ligation and transformation looks bad, transform the 4C and 16C ligations - Mike</li>
 
-
<li>
 
-
When primers arrive, PCR out mCherry from JMIL intein plasmid, PCR out INPNC, then assembly PCR for INPNC-mCherry</li>
 
-
</ul>
 
-
<br>
 
-
 
-
 
-
<p><b>August 30</b></p>
 
-
<ul>
 
-
<li>
 
-
Protein purification of His-DARPin-HA
 
-
</li>
 
-
<li>
 
-
PCR purify INPNC-mCherry, run a few ul on a diagnostic gel ? results call for gel purifiation ? Gel purify INPNC-mCherry ? yield was bad, redo PCR  ? gel purify
 
-
</li>
 
-
<li>
 
-
Digest INPNC-mCherry and pET26b with NdeI and HindIII, ligate at RT for 1 hr (and maybe some at 4C or 16C overnight), and transform into pET26b
 
-
</li>
 
-
<li>
 
-
Start DH5a cultures for INPNC, INPNC-HA, His-DARPin-HA, pET-ClyA-His, pET-PelB-ClyA-His and pDawn-ClyA-His for minipreps
 
-
</li>
 
-
<li>
 
-
Repick colonies of pBAD33-eGFP from 16C or 4C plate
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
                   
 
-
  </div>
 
-
 
-
</div>
 
-
 
-
 
-
 
-
<p style="text-align:center;color:white;">September 2012 Notebook</p>
 
-
<div id="accordion-container">
 
-
<h2 class="accordion-header">Week 13</h2>
 
-
<div class="accordion-content">
 
-
 
-
                        </div>
 
-
 
-
<h2 class="accordion-header">Week 14</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
<p><b>September 10</b></p>
 
-
<ul>
 
-
<li>
 
-
Mike - pick INPNC-mCherry colony (transformed Sample #2) and split into + and - cultures
 
-
</li>
 
-
<li>Start BL21 cultures of INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin-HA, His-Intein-mCherry
 
-
</li>
 
-
<li>
 
-
Peter - Protein purification of His-DARPin-HA, ClyA-His
 
-
</li>
 
-
<li>
 
-
Avin - Dilute (9am) &amp; Induce (?) His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA at 0.5
 
-
</li>
 
-
<li>
 
-
Ashwin - Wiki work
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>September 11</b></p>
 
-
<ul>
 
-
<li>
 
-
Mike - Make glycerol stock of INPNC-mCherry, Dilute and induce INPNC-mCherry, INPNC, INPNC-HA, DARPin-HA, INPNC-DARPin HA, His-Intein-mCherry
 
-
</li>
 
-
<li>
 
-
Peter - Make electro/chemically competent cells, transform
 
-
</li>
 
-
<li>
 
-
Ashwin - Pick up S. typhimurium from Mark Goulian
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>September 12</b></p>
 
-
<ul>
 
-
<li>
 
-
Peter - Lysis and spin down (for INPNC-mCherry, aliquot  1mL of both (+) and (-) for Avin
 
-
</li>
 
-
<li>Peter- spin down and check if red, then lyse) , put in 4C, check Nissle eGFP colonies
 
-
</li>
 
-
<li>
 
-
Avin - Immuno experiment on His-DARPin-HA, INPNC-HA, INPNC-DARPin-HA, Take INPNC-mCherry, His-Intein-mCherry, fix and mount onto scope slides
 
-
</li>
 
-
</ul>
 
-
</br>
 
-
 
-
<p><b>September 13</b></p>
 
-
<ul>
 
-
<li>
 
-
Mike - Bradford assay, Run protein gel</li>
 
-
<li>Peter - </li>
 
-
<li>
 
-
Avin - Observe BL21 transformatons under confocal
 
-
</li>
 
-
<li>
 
-
Ashwin - colony PCR on S. typhimurium for MisL
 
-
</li>
 
-
</ul>
 
-
<br>
 
-
 
-
<p><b>September 14</b></p>
 
-
<ul>
 
-
<li>
 
-
Mike -
 
-
</li>
 
-
<li>
 
-
Peter -
 
-
</li>
 
-
<li>
 
-
Avin - core confocal?
 
-
</li>
 
-
<li>
 
-
Ashwin -
 
-
</li>
 
-
</ul>
 
-
 
-
&nbsp;&nbsp;<p>Other<p>
 
-
<ul>
 
-
<li>Order scfv plasmid, scfv primers</li>
 
-
<li>
 
-
Order MisL Primers, pick up misL, PCR on S. typhimurium
 
-
</li>
 
-
<li>
 
-
Chemical/Electrocompetent Nissle
 
-
</li>
 
-
<li>
 
-
Make LB, Glycerol
 
-
</li>
 
-
<li>
 
-
Wiki
 
-
</li>
 
-
</ul>
 
-
</br>
 
-
                        </div>
 
-
 
-
<h2 class="accordion-header">Week 15</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
                        </div>
 
-
 
-
<h2 class="accordion-header">Week 16</h2>
 
-
 
-
<div class="accordion-content">
 
-
 
-
                        </div>
 
-
 
</div>
</div>

Revision as of 15:34, 26 June 2013

Using TimelineJS: Last year's notebook:

June 2012 Notebook

Week 1

June 6th

  • Set up some lab equipment
  • Autoclaved for a while
  • Organized biobrick stuff
  • Called Vinoo about DNA planning

June 7th

  • Transformed Cph8, pLsr, and LuxS
  • Placed order with Vinoo
  • Developed idea using PGY/PCN system to activate a gene

Week 2

June 11th

 

Wet Lab

  • PCR'd mCherry from NAS157
  • Ran 1% Gel and purified product
  

Dry Lab

  • Designed primers for LsR promoter
  • Meeting with Dr. Sarkar

June 12th

 

Wet Lab

  • Digested mCherry PCR product with BamHI and NotI
  • Column purified mCherry and ligated into NAS152 backbone
  • Transformed NAS152-mCherry into DH5alpha
  • Poured 25 LB-Kan plates
  

Dry Lab

  • Research more information about bacterial drug delivery system
  • More research into biofilm project

June 14th

  

Dry Lab

  • Met with Dr. Goulian, obtained pDawn and pDusk
  • Identified inaK as a surface display gene we can use

Week 3

June 18th

  

Wet Lab

  • Miniprep pDawn and pDusk
  • Test cut pDawn and pDusk with XmaI, analytical gel was correct
  • Prep cut pDawn and pDusk with BamHI and NotI, gel purified
  

Dry Lab

  • Ordered and picked up PCR purification kit from cell center
  • Additional orders through cell center
  • Designed primers for one of Peter's components (forgot which)

June 20

  

Wet Lab

  • Picked 2 colonies of pDawn-mCherry, innoculated in 5 mL of LB and 50 ug/mL of Kan
  • PCR purified fragments (Peter), then ran gel?
  

Dry Lab

  • Researched DARPin binding domains and linkers
  • Finalized some biobrick orders
  • Finalized synthesis order (minus linker)

Retrieved from "http://2013.igem.org/Team:UPenn/Notebook"