Difference between revisions of "Team:SPSingapore/Notebook-Week-4"

 
(20 intermediate revisions by the same user not shown)
Line 22: Line 22:
 
align:center;
 
align:center;
 
}
 
}
−
.nbtable{background:transparent;border-collapse: collapse; border: 1px solid black;}
 
−
.nbtable td{padding-left:15px;padding-right:15px;padding-top:3px;padding-bottom:3px;}
 
  
 
h1 {
 
h1 {
Line 66: Line 64:
 
<div id='cssmenu'>
 
<div id='cssmenu'>
 
<ul>
 
<ul>
−
  <li><a href='https://2015.igem.org/Team:SPSingapore/'><span>Home</span></a></li>
+
<li class = 'active'><a href='https://2015.igem.org/Team:SPSingapore/'><span>Home</span></a></li>
−
  <li><a href='https://2015.igem.org/Team:SPSingapore/Team'><span>Team</span></a></li>
+
<li><a href='https://2015.igem.org/Team:SPSingapore/Team'><span>Team</span></a>
−
  <li><a href='https://2015.igem.org/Team:SPSingapore/Project'><span>Project</span></a></li>
+
<ul>
−
  <li><a href='https://2015.igem.org/Team:SPSingapore/Protocol'><span>Protocol</span></a></li>
+
<li><a href="https://2015.igem.org/Team:SPSingapore/Team">Overview</a></li>
−
  <li><a href='https://2015.igem.org/Team:SPSingapore/Parts'><span>Parts</span></a></li>
+
<li><a href="https://igem.org/Team.cgi?id=1804">Official Profile</a></li>
−
  <li class = 'active'><a href='https://2015.igem.org/Team:SPSingapore/Notebook'><span>Notebook</span></a></li>
+
        <li><a href="https://2015.igem.org/Team:SPSingapore/Team Bios">Team Bios</a></li>
−
  <li><a href='https://2015.igem.org/Team:SPSingapore/Practices'><span>Human Practices</span></a></li>
+
        <li><a href="https://2015.igem.org/Team:SPSingapore/Mentors">Mentors</a></li>
−
  <li class='last'><a href='https://2015.igem.org/Team:SPSingapore/Safety'><span>Safety</span></a></li>
+
        <li><a href="https://2015.igem.org/Team:SPSingapore/Attributions">Attributions</a></li>
 +
</ul>
 +
</li>
 +
<li><a href='https://2015.igem.org/Team:SPSingapore/Project'><span>Project</span></a>
 +
<ul>
 +
<li><a href='https://2015.igem.org/Team:SPSingapore/Project'>Overview</a></li>
 +
        <li><a href="https://2015.igem.org/Team:SPSingapore/Invasin">Invasin + Listerolysin</a></li>
 +
        <li><a href="https://2015.igem.org/Team:SPSingapore/ESAQS">esa Quorum Sensing</a></li>
 +
        <li><a href="https://2015.igem.org/Team:SPSingapore/Anaerobic Promoter">Anaerobic Promoter</a></li>
 +
        <li><a href="https://2015.igem.org/Team:SPSingapore/Parts">Parts</a></li>
 +
</ul>
 +
</li>
 +
<li><a href='https://2015.igem.org/Team:SPSingapore/Notebook'><span>Notebook</span></a>
 +
<ul>
 +
<li><a href="https://2015.igem.org/Team:SPSingapore/Protocol">Protocols</a></li>
 +
        <li><a href="https://2015.igem.org/Team:SPSingapore/Notebook">Entries</a></li>
 +
</ul>
 +
</li>
 +
<li><a href='https://2015.igem.org/Team:SPSingapore/Practices'><span>Human Practices</span></a>
 +
<ul>
 +
<li><a href="https://2015.igem.org/Team:SPSingapore/Practices">Overview</a></li>
 +
<li><a href="https://2015.igem.org/Team:SPSingapore/Workshop">Workshop</a></li>
 +
<li><a href="https://2015.igem.org/Team:SPSingapore/Workshop Materials">Workshop Materials</a></li>
 +
        <li><a href="https://2015.igem.org/Team:SPSingapore/Interview">Consultations</a></li>
 +
</ul>
 +
</li>
 +
<li><a href='https://2015.igem.org/Team:SPSingapore/Safety'><span>Safety</span></a></li>
 +
<li class='last'><a href='https://2015.igem.org/Team:SPSingapore/Medals'><span>Medals</span></a></li>
 
</ul>
 
</ul>
 
</div>
 
</div>
Line 79: Line 104:
 
<div id='sidemenu' style = "float:left">
 
<div id='sidemenu' style = "float:left">
 
<ul>
 
<ul>
 +
  <li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook"><span>NOTEBOOK</span></a>
 +
  <ul><li class = 'last'>&nbsp;</li></ul></li>
 +
 
 +
  <li><a href = "https://2015.igem.org/Team:SPSingapore/Protocol"><span>PROTOCOL</span></a>
 +
  <ul><li class = 'last'>&nbsp;</li></ul></li>
 +
 
   <li class='last'><a href = "https://2015.igem.org/Team:SPSingapore/Notebook"><span>ENTRIES</span></a>
 
   <li class='last'><a href = "https://2015.igem.org/Team:SPSingapore/Notebook"><span>ENTRIES</span></a>
 
<ul>
 
<ul>
Line 97: Line 128:
 
         <li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-15"><span>Week 15 (30/8 - 5/9)</span></a></li>
 
         <li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-15"><span>Week 15 (30/8 - 5/9)</span></a></li>
 
         <li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-16"><span>Week 16 (6/9 - 12/9)</span></a></li>
 
         <li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-16"><span>Week 16 (6/9 - 12/9)</span></a></li>
−
         <li class = 'last'><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-17"><span>Week 17 (13/8 - 17/9)</span></a></li>
+
         <li class = 'last'><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-17"><span>Week 17 (13/9 - 18/9)</span></a></li>
 
       </ul>
 
       </ul>
 
   </li>
 
   </li>
Line 127: Line 158:
 
<!----------------------- Entry Start ------------------------>
 
<!----------------------- Entry Start ------------------------>
 
<center>
 
<center>
−
<table style = "width:700px; border-collapse: collapse;">
+
<table style = "width:750px; border-collapse: collapse;">
  
−
<tr class = "tablenotebook"><td><p class = "paper">
+
<! ------------------------------- new day ----------------------------->
−
14th June 2015
+
 
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; June 14
 
<br><br>
 
<br><br>
−
<strong>Yi Han</strong>
+
<span class = "brown"> Anaerobic Promoter</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 
<br><br>
 
<br><br>
−
&bull; Miniprep of gfp plasmids, preparation of samples for sequencin
+
<div class = "divnbcontent">
−
<br>
+
<span class = "nbcontent">
−
</p>
+
PCR
 +
<br> &#9654; RP_XhoI_GFP and FP_KpnI_GFP with GFP-3 as template
 +
<br> &#9654; for 7 reactions with control
 +
</span><br>
 +
 
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td colspan = 2><b>Mastermix * 8</b></td></tr>
 +
<tr><td>PCR buffer  </td><td>80ul</td></tr>
 +
<tr><td>Primers  </td><td>0.8ul/0.8ul</td></tr>
 +
<tr><td>DNA polymerase  </td><td>4ul</td></tr>
 +
<tr><td>dH2O  </td><td>494ul</td></tr>
 +
<tr><td>Templates  </td><td>38.75</td></tr>
 +
</table>
 +
<br><br>
 +
 
 +
<span class = "nbcontent">
 +
Digest more plasmid (Esa plasmid)
 +
</span><br>
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td>4 rxns ( KpnI/XhoI digest) </td><td>50ul each</td></tr
 +
<tr><td> Buffer </td><td> 20ul </td></tr>
 +
<tr><td> KpnI </td><td> 2ul </td></tr>
 +
<tr><td> XhoI 2ul </td><td> </td></tr>
 +
<tr><td> DNA </td><td> 34.8ul </td></tr>
 +
<tr><td> H20 </td><td> 141.2ul </td></tr>
 +
</table>
 +
 
 +
</div>
 +
<br><br>
 +
 
 +
 
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
  
−
<tr class = "tablenotebook"><td><p class = "paper">
+
 
−
9th June 2015
+
<! ------------------------------- new day ----------------------------->
 +
 
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; June 15
 
<br><br>
 
<br><br>
−
<strong>Yi Han</strong>
+
<span class = "brown"> Anaerobic Promoter</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yun Ting &#9728;</font>
 
<br><br>
 
<br><br>
−
&bull; All gfp plasmids had a correct sequence<br>
+
<div class = "divnbcontent">
−
&bull; Yun Ting kept glycerol stocok for all, and inoculation of 3mL of gfp3 into 100mL LB+amp for midiprep<br>
+
<span class = "nbcontent">
−
</p>
+
PCR: RP_XhoI_GFP and FP_KpnI_GFP with GFP midiprep for 15 reactions with control:
 +
</span><br>
 +
 
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> PCR buffer </td><td> 170ul </td></tr>
 +
<tr><td> Primers </td><td> 1.7ul, 1.7ul </td></tr>
 +
<tr><td> dNTP </td><td> 34 ul </td></tr>
 +
<tr><td> DNA polymerase </td><td> 8.5 ul </td></tr>
 +
<tr><td> dH2O </td><td> 1127.1ul </td></tr>
 +
<tr><td> Templates </td><td> 17ul </td></tr>
 +
<tr><td> Total </td><td> 1360ul </td></tr>
 +
</table>
 +
<br>
 +
<span class = "nbcontent">
 +
PCR protocol “GFP 1” (32 cycles)
 +
</span>
 +
 
 +
</div>
 +
 
 +
<br><br>
 +
<span class = "brown"> Anaerobic Promoter</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
GFP PCR product -> 448ng/ul
 +
<br> Gel extraction
 +
<br> Qiagen gel extraction kit at MBI
 +
<br> Esa gel band from 14/6
 +
<br> Nanodrop: 16.4 ng/ul, 260/280 = 2.60
 +
</span><br>
 +
<br>
 +
 
 +
<span class = "nbcontent">
 +
Plasmid construction
 +
</span><br>
 +
<span class = "nbcontent">
 +
RE digest (KpnI, XhoI) 3 Replicates of the following:
 +
</span><br>
 +
 
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> DNA </td><td> 2.6ul </td></tr>
 +
<tr><td> Buffer </td><td> 5ul </td></tr>
 +
<tr><td> H2O </td><td> 41.5ul </td></tr>
 +
<tr><td> KpnI </td><td> 0.5ul </td></tr>
 +
<tr><td> XhoI </td><td> 0.5ul </td></tr>
 +
</table>
 +
<br><br>
 +
 
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td>
 +
Ligation (2x reaction)</td><td>
 +
40ul</td></tr>
 +
 
 +
<tr><td>
 +
10X T4 DNA ligase buffer</td><td>
 +
4ul</td></tr>
 +
 
 +
<tr><td>
 +
Vector DNA (16ng/ul)</td><td>
 +
100ng -> 6.25ul</td></tr>
 +
 
 +
<tr><td>
 +
insert DNA (75ng)</td><td>
 +
12 ul </td></tr>
 +
 +
<tr><td>
 +
T4 DNA Ligase </td><td>
 +
2ul</td></tr>
 +
 
 +
<tr><td>
 +
H2O</td><td>
 +
16ul</td></tr>
 +
</table>
 +
<br><br>
 +
 
 +
<span>
 +
&#9654; Note: ALL digested DNA in tube labeled lacGFP.ligation was used
 +
<br> &#9654; Nanodrop of ligation: 1114.0ng/ul. 260/280 = 3.7
 +
</span>
 +
 
 +
 
 +
</div>
 +
 
 +
<br><br>
 +
 
 +
 
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
  
−
<tr class = "tablenotebook"><td><p class = "paper">
+
 
−
10th June 2015
+
<! ------------------------------- new day ----------------------------->
 +
 
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; June 16
 
<br><br>
 
<br><br>
−
<strong>Yi Han & Yun Ting</strong>
+
<span class = "brown"> Anaerobic Promoter</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 
<br><br>
 
<br><br>
−
&bull; Midiprep of gfp plasmid  
+
<div class = "divnbcontent">
−
<br>
+
<span class = "nbcontent">
−
&bull; PCR of gfp with KpnI-gfp and XhoI-gfp primers
+
Transformation of ligated esa-GFP plasmid into DH5\alpha cells
−
</p>
+
</span><br>
 +
<span class = "nbcontent">
 +
DH5alpha, unlabelled brown vial inside DH5\alpha box at -80
 +
<br> &#9654; Plates spread at 1140h
 +
<br> &#9654; LB + chloroamphenicol
 +
<br> &emsp;&emsp;&emsp;&emsp; 4x (100ul)
 +
<br> &emsp;&emsp;&emsp;&emsp; 10x (40ul)
 +
<br> &emsp;&emsp;&emsp;&emsp; 20x (20ul)
 +
<br> &emsp;&emsp;&emsp;&emsp; LB - 20x (20ul)
 +
</span><br>
 +
<span class = "nbcontent">
 +
Remaining transformed cells (~220ul) are kept at 4C
 +
<br> &#9654; labeled as placGFP in brown tube
 +
</span>
 +
 
 +
 
 +
</div>
 +
 
 +
<br><br>
 +
 
 +
 
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
  
−
<tr class = "tablenotebook"><td><p class = "paper">
+
<! ------------------------------- new day ----------------------------->
−
11th June 2015
+
 
 +
 
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; June 17
 
<br><br>
 
<br><br>
−
<strong>Yun Ting & Duy</strong>
+
<span class = "brown"> Anaerobic Promoter</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 
<br><br>
 
<br><br>
−
&bull; Nanodrop of gfp product &rarr; 595.5ng/ul
+
<div class = "divnbcontent">
−
<br>
+
<span class = "nbcontent">
−
&bull; RE digest of EsaR vector with KpnI/XHoI<br>
+
only 4x placGFP plate has colonies (7)
−
&bull; Gel electrophoreseis at 1000V for 30min<br>
+
<br> &#9654; spread one new LB + chlor plate with 200ul of transformed bacteria
−
&bull; Gel extraction: 3.9ng/ul and 6.9ng/ul for Esa fragment and GFP --&gt; low yield<br>
+
<br> &#9654; picked 6 colonies to grow for miniprep in LB + chl liq media
−
</p>
+
<br> &#9654; Conclusion: Protocol works but optimization for increase vol needed
 +
</span><br>
 +
<span class = "nbcontent">
 +
Cloning of synparts in amp vector
 +
<br> &#9654; comes as 4mg dry DNA
 +
<br> &#9654; +40ul of DI/RNAse free H20
 +
<br> &#9654; for transformation, 150ml wed
 +
</span><br><br>
 +
<span class = "nbcontent">
 +
Miniprep of placGFP followed by RE digest (Kpn, XhoI): 50 ul total
 +
</span><br>
 +
 
 +
<table class = "nbtable" style = "margin-left:50px">
 +
<tr><td> Buffer </td><td> 2.5 </td></tr>
 +
<tr><td> KpnI </td><td> 1 </td></tr>
 +
<tr><td> XhoI </td><td> 1 </td></tr>
 +
<tr><td> DNA </td><td> 10 </td></tr>
 +
<tr><td> H20 </td><td> 120.25 </td></tr>
 +
</table>
 +
<br><br>
 +
 
 +
<span class = "nbcontent">
 +
Colony PCR for synparts
 +
<br> &#9654; Failed
 +
<br> &#9654; No specific bands produced
 +
<br> &#9654; Might need optimization
 +
</span>
 +
 
 +
</div>
 +
 
 +
<br><br>
 +
 
 +
<span class = "blue"> Invasin + Listerolysin</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yan Ting &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
 
 +
<span class = "nbcontent">
 +
Subculture of HEK 293
 +
<br> &#9654; P4 -> p5
 +
<br> &#9654; grow/split into 150mm dish for freezing on sat (20/6)
 +
<br> &emsp;&emsp;&emsp;&emsp; 30ml DMEM + 1ml cells
 +
<br> &#9654; 90mm dish for maintenance (buffer)
 +
<br> &emsp;&emsp;&emsp;&emsp; 10ml DMEM + 30ul cells
 +
<br> &#9654; Cells combined from 3 90mm dishes
 +
</span>
 +
</div>
 +
 
 +
<br><br>
 +
 
 +
 
 +
 
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
  
−
<tr class = "tablenotebook"><td><p class = "paper">
+
 
−
12th June 2015
+
<! ------------------------------- new day ----------------------------->
 +
 
 +
 
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; June 19
 
<br><br>
 
<br><br>
−
<strong>Yun Ting</strong>
+
<span class = "black"> esa Quorum Sensing</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 
<br><br>
 
<br><br>
−
&bull; Gel extraction using Promega binding solution to melt gel, followed by thermo scientific kit
+
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
RE digest (XhoI, KpnI) of esa and GFP
 +
</span><br>
 +
 
 +
</div>
 +
 
 
<br><br>
 
<br><br>
−
<strong>Adrian</strong>
+
 
 +
<span class = "black"> esa Quorum Sensing</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yun Ting &#9728;</font>
 
<br><br>
 
<br><br>
−
&bull; Gel extraction optimisation
+
<div class = "divnbcontent">
−
<br>
+
<span class = "nbcontent">
−
&bull; Hypothesised that the Binding buffer has a problem/DNA does not bind to column
+
Gel electrophoresis (100V, 40min).
−
<br>
+
<br>&#9654; Lane 2: gpf: no bands
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
<br>&#9654; Lane 3: 100bp ladder
−
1: 2X promega binding buffer volume<br>
+
<br>&#9654; Lane 4: blank
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
<br>&#9654; Lane 5: esa: 2 bands
−
2: Increase incubation time for binding to 5min
+
<br>&#9654; Lane 6: 1kb ladder
−
<br>
+
<br>&#9654; Lane 7:blank
−
&bull; Switch binding buffer to that of thermo scientific PCR purification kit<br>
+
</span>
−
&bull; Use sodium acetate if available? To facilitate stronger binding to column
+
 
−
<br>
+
</div>
−
<u>Results:</u><br>
+
 
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1: ~10ng/ul<br>
+
<br><br>
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2: ~9ng/ul<br>
+
 
−
&bull;Further optimisation-&gt; warm buffer, incubate for 5min<br>
+
 
−
&bull;Elute in 30/20ul smaller volumes<br>
+
</div>
−
</p>
+
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
  
−
<tr class = "tablenotebook"><td><p class = "paper">
+
<! ------------------------------- new day ----------------------------->
−
13th June 2015
+
 
 +
 
 +
 
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; June 20
 
<br><br>
 
<br><br>
−
<strong>Yi Han</strong>
+
<span class = "blue"> Invasin + Listerolysin</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yan Ting &#9728;</font>
 
<br><br>
 
<br><br>
−
1: Thermoscientific miniprep columns with 2XThermoscientific binding buffer
+
<div class = "divnbcontent">
−
<br>
+
<span class = "nbcontent">
−
2: Thermoscientific PCR purification kit 2X buffer
+
Freezing of HEK293
−
<br>
+
<br> &#9654; P6
−
3: Promega kit 2X buffer
+
<br> &#9654; cells from a 150 mm dish and 90mm dish
−
<br><br><br>
+
</span>
−
</p>
+
 
 +
</div>
 +
 
 +
<br><br>
 +
 
 +
 
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
 +
 +
 +
 +
<! ------------------------------- new day ----------------------------->
 +
 +
  
 
</table>
 
</table>
Line 235: Line 516:
  
 
<! ------------------------------- Footer START ------------------------------------------------>
 
<! ------------------------------- Footer START ------------------------------------------------>
 +
  
 
<div id="footer1">
 
<div id="footer1">
Line 240: Line 522:
 
<div class="left_sec">
 
<div class="left_sec">
 
<p>
 
<p>
−
<img src = "https://static.igem.org/mediawiki/2015/a/ab/SPSingapore_Team-Logo-New.png" height = "80px">
+
<img src = "https://static.igem.org/mediawiki/2015/a/ab/SPSingapore_Team-Logo-New.png" height = "60px" style="margin-top:5px;margin-left:-130px;">
 
</p>
 
</p>
 
</div>
 
</div>
 
<div class="right_sec">
 
<div class="right_sec">
 
<p>
 
<p>
−
<img src = "https://static.igem.org/mediawiki/2015/0/05/SPSingapore_NUS-Logo.png" height = "60px" style = "margin-top:10px;margin-right:30px;padding-right:30px;border-right: 3px solid lightgrey;">
+
<img src="https://static.igem.org/mediawiki/2015/0/05/SPSingapore_NUS-Logo.png" height="50px" style="margin-top:10px;margin-right:30px;padding-right:30px;border-right: 3px solid lightgrey;">
−
<img src = "https://static.igem.org/mediawiki/2015/7/72/SPSingapore_SPS-Logo.png" height = "60px" style = "margin-top:10px;margin-right:50px;"></a>
+
<img src="https://static.igem.org/mediawiki/2015/7/72/SPSingapore_SPS-Logo.png" height="50px" style="margin-top:10px;margin-right:30px;">
 
</p>
 
</p>
 
</div>
 
</div>

Latest revision as of 23:16, 18 September 2015


Research Notebook

Week 4 (14/6 - 20/6)

▪ June 14

Anaerobic Promoter    ☀ Yi Han ☀

PCR
▶ RP_XhoI_GFP and FP_KpnI_GFP with GFP-3 as template
▶ for 7 reactions with control
Mastermix * 8
PCR buffer 80ul
Primers 0.8ul/0.8ul
DNA polymerase 4ul
dH2O 494ul
Templates 38.75


Digest more plasmid (Esa plasmid)
4 rxns ( KpnI/XhoI digest) 50ul each
Buffer 20ul
KpnI 2ul
XhoI 2ul
DNA 34.8ul
H20 141.2ul


▪ June 15

Anaerobic Promoter    ☀ Yun Ting ☀

PCR: RP_XhoI_GFP and FP_KpnI_GFP with GFP midiprep for 15 reactions with control:
PCR buffer 170ul
Primers 1.7ul, 1.7ul
dNTP 34 ul
DNA polymerase 8.5 ul
dH2O 1127.1ul
Templates 17ul
Total 1360ul

PCR protocol “GFP 1” (32 cycles)


Anaerobic Promoter    ☀ Yi Han ☀

GFP PCR product -> 448ng/ul
Gel extraction
Qiagen gel extraction kit at MBI
Esa gel band from 14/6
Nanodrop: 16.4 ng/ul, 260/280 = 2.60

Plasmid construction
RE digest (KpnI, XhoI) 3 Replicates of the following:
DNA 2.6ul
Buffer 5ul
H2O 41.5ul
KpnI 0.5ul
XhoI 0.5ul


Ligation (2x reaction) 40ul
10X T4 DNA ligase buffer 4ul
Vector DNA (16ng/ul) 100ng -> 6.25ul
insert DNA (75ng) 12 ul
T4 DNA Ligase 2ul
H2O 16ul


▶ Note: ALL digested DNA in tube labeled lacGFP.ligation was used
▶ Nanodrop of ligation: 1114.0ng/ul. 260/280 = 3.7


▪ June 16

Anaerobic Promoter    ☀ Yi Han ☀

Transformation of ligated esa-GFP plasmid into DH5\alpha cells
DH5alpha, unlabelled brown vial inside DH5\alpha box at -80
▶ Plates spread at 1140h
▶ LB + chloroamphenicol
     4x (100ul)
     10x (40ul)
     20x (20ul)
     LB - 20x (20ul)
Remaining transformed cells (~220ul) are kept at 4C
▶ labeled as placGFP in brown tube


▪ June 17

Anaerobic Promoter    ☀ Yi Han ☀

only 4x placGFP plate has colonies (7)
▶ spread one new LB + chlor plate with 200ul of transformed bacteria
▶ picked 6 colonies to grow for miniprep in LB + chl liq media
▶ Conclusion: Protocol works but optimization for increase vol needed
Cloning of synparts in amp vector
▶ comes as 4mg dry DNA
▶ +40ul of DI/RNAse free H20
▶ for transformation, 150ml wed

Miniprep of placGFP followed by RE digest (Kpn, XhoI): 50 ul total
Buffer 2.5
KpnI 1
XhoI 1
DNA 10
H20 120.25


Colony PCR for synparts
▶ Failed
▶ No specific bands produced
▶ Might need optimization


Invasin + Listerolysin    ☀ Yan Ting ☀

Subculture of HEK 293
▶ P4 -> p5
▶ grow/split into 150mm dish for freezing on sat (20/6)
     30ml DMEM + 1ml cells
▶ 90mm dish for maintenance (buffer)
     10ml DMEM + 30ul cells
▶ Cells combined from 3 90mm dishes


▪ June 19

esa Quorum Sensing    ☀ Yi Han ☀

RE digest (XhoI, KpnI) of esa and GFP


esa Quorum Sensing    ☀ Yun Ting ☀

Gel electrophoresis (100V, 40min).
▶ Lane 2: gpf: no bands
▶ Lane 3: 100bp ladder
▶ Lane 4: blank
▶ Lane 5: esa: 2 bands
▶ Lane 6: 1kb ladder
▶ Lane 7:blank


▪ June 20

Invasin + Listerolysin    ☀ Yan Ting ☀

Freezing of HEK293
▶ P6
▶ cells from a 150 mm dish and 90mm dish