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

 
(15 intermediate revisions by the same user not shown)
Line 64: 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 77: 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 95: 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 119: Line 152:
 
<table><tr><td>
 
<table><tr><td>
 
<div style = "border-top: 5px solid blue; width:750px; text-align:justify;margin-bottom: 20px; line-height:normal;">
 
<div style = "border-top: 5px solid blue; width:750px; text-align:justify;margin-bottom: 20px; line-height:normal;">
−
<h3 style ="margin-left:20px;">Week 1 (24/5 - 30/5)</h3>
+
<h3 style ="margin-left:20px;">Week 4 (14/6 - 20/6)</h3>
  
  
Line 126: Line 159:
 
<center>
 
<center>
 
<table style = "width:750px; border-collapse: collapse;">
 
<table style = "width:750px; border-collapse: collapse;">
 +
 +
<! ------------------------------- new day ----------------------------->
  
 
<tr class = "tablenotebook"><td><div class = "paper">
 
<tr class = "tablenotebook"><td><div class = "paper">
−
May 25
+
&#9642; June 14
 
<br><br>
 
<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>
 
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 
<br><br>
 
<br><br>
−
<ul>
+
<div class = "divnbcontent">
−
<li>&#10097;&#10097;&#10097; Received 2 bacterial stab cultures, EsaR/I plasmid from addgene (CHL) and BBa_K299812 from iGEM HQ.</li>
+
<span class = "nbcontent">
−
<li>&#10097;&#10097;&#10097; Streaked out on plates with amp./li>
+
PCR
−
</ul>
+
<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>
 
<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>
 
<div style = "border-top: 2px solid turquoise;"></div>
 
<div style = "border-top: 2px solid turquoise;"></div>
Line 143: Line 207:
 
</tr>
 
</tr>
  
 +
 +
<! ------------------------------- new day ----------------------------->
  
 
<tr class = "tablenotebook"><td><div class = "paper">
 
<tr class = "tablenotebook"><td><div class = "paper">
−
May 26
+
&#9642; June 15
 
<br><br>
 
<br><br>
−
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Adrian &#9728;</font>
+
<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>
−
&#10097;&#10097;&#10097; Transformed:<br>
+
<div class = "divnbcontent">
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;+Kit plate 1 9N Ba_K763002 chl<br>
+
<span class = "nbcontent">
−
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;+Kit plate 4 13L BBa_E0040 amp
+
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>
 
<br>
−
&#10097;&#10097;&#10097; DNa was received in powder form in plates, and resuspended in 10ul ultrapure H2O respectively. Plates were stored in -20/
+
<span class = "nbcontent">
 +
PCR protocol “GFP 1” (32 cycles)
 +
</span>
 +
 
 +
</div>
 +
 
 
<br><br>
 
<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>
 
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 
<br><br>
 
<br><br>
−
&#10097;&#10097;&#10097; Plates cracked in incubator as they dried from lack of humidity.
+
<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>
 
<br>
−
&#10097;&#10097;&#10097; Transfer to small incubator with beaker of water for humidity no single colonies for inv plasmid -> streak again.
+
 
−
<br>
+
<span class = "nbcontent">
−
&#10097;&#10097;&#10097; Wrong antibiotic for EsaR/I plasmid &rarr; streak out again
+
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>
 
<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>
 
<div style = "border-top: 2px solid turquoise;"></div>
 
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
 +
 +
 +
<! ------------------------------- new day ----------------------------->
  
 
<tr class = "tablenotebook"><td><div class = "paper">
 
<tr class = "tablenotebook"><td><div class = "paper">
−
May 28
+
&#9642; June 16
 
<br><br>
 
<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>
 
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 
<br><br>
 
<br><br>
−
&#10097;&#10097;&#10097; Inoculate single colony of invasin plasmid carrying bacteria in 3mL LB+amp
+
<div class = "divnbcontent">
−
<br>
+
<span class = "nbcontent">
−
&#10097;&#10097;&#10097; Transformation of 13L repeated with 1ul of DNA.
+
Transformation of ligated esa-GFP plasmid into DH5\alpha cells
 +
</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>
 
</div>
 +
 
<br><br>
 
<br><br>
 +
 +
 +
</div>
 
<div style = "border-top: 2px solid turquoise;"></div>
 
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
 +
 +
<! ------------------------------- new day ----------------------------->
 +
  
 
<tr class = "tablenotebook"><td><div class = "paper">
 
<tr class = "tablenotebook"><td><div class = "paper">
−
May 29
+
&#9642; June 17
 
<br><br>
 
<br><br>
−
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Xin Yi &#9728;</font>
+
<span class = "brown"> Anaerobic Promoter</span>
−
<br><br>
+
&emsp;&emsp;
−
&#10097;&#10097;&#10097; No colonies grew for 13L on all plates &rarr; adjust incubator, make new media
+
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 
<br><br>
 
<br><br>
−
&#10097;&#10097;&#10097; Miniprep of inoculated bacteria for invasin plasmid, incubate sample in 37degC for 1h 20min:
+
<div class = "divnbcontent">
−
<br>
+
<span class = "nbcontent">
−
<table class = "nbtable" style = "margin-left:100px;">
+
only 4x placGFP plate has colonies (7)
−
<tr><td><b>RE of invasin plasmid</b></td>
+
<br> &#9654; spread one new LB + chlor plate with 200ul of transformed bacteria
−
<td><b>RE control</b></td></tr>
+
<br> &#9654; picked 6 colonies to grow for miniprep in LB + chl liq media
−
<tr><td>Rsal 1ul</td>
+
<br> &#9654; Conclusion: Protocol works but optimization for increase vol needed
−
<td>Rsal 0 ul</td></tr>
+
</span><br>
−
<tr><td>EcoRI 1 ul</td>
+
<span class = "nbcontent">
−
<td>EcoRI 0 ul</td></tr>
+
Cloning of synparts in amp vector
−
<tr><td>INv plasmid 7.5ul</td>
+
<br> &#9654; comes as 4mg dry DNA
−
<td>Inv plasmid 7.5 ul</td></tr>
+
<br> &#9654; +40ul of DI/RNAse free H20
−
<tr><td>H2O 35.5 ul</td>
+
<br> &#9654; for transformation, 150ml wed
−
<td>H2O 37.5 ul</td></tr>
+
</span><br><br>
−
<tr><td>NEB buffer 4.5 ul</td>
+
<span class = "nbcontent">
−
<td>Buffer 4.5 ul</td></tr>
+
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>
 
</table>
−
<br>
 
−
<div>&#10097;&#10097;&#10097; Repeat transformation of 13L with 1ul</div>
 
 
<br><br>
 
<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>
 
<div style = "border-top: 2px solid turquoise;"></div>
 
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
 +
 +
 +
<! ------------------------------- new day ----------------------------->
 +
  
 
<tr class = "tablenotebook"><td><div class = "paper">
 
<tr class = "tablenotebook"><td><div class = "paper">
−
May 30
+
&#9642; June 19
 
<br><br>
 
<br><br>
−
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Xin Yi & Yi Han &#9728;</font>
+
<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>
−
&#10097;&#10097;&#10097; Miniprep of EsaR/I plasmid for 4 colonies
+
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
RE digest (XhoI, KpnI) of esa and GFP
 +
</span><br>
 +
 
 +
</div>
 +
 
 
<br><br>
 
<br><br>
−
&#10097;&#10097;&#10097; Restriction digest for EsaR with XbaI/BamHI:
+
 
 +
<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>
−
<span><b><u>RE reaction</u></b></span><br>
+
<div class = "divnbcontent">
−
<span>5ul plasmid</span><br><span>5ul buffer</span><br><span>1ul XbaI</span><br><span>1ul BamHI</span><br><span>Add H2O to 50ul</span>
+
<span class = "nbcontent">
 +
Gel electrophoresis (100V, 40min).
 +
<br>&#9654; Lane 2: gpf: no bands
 +
<br>&#9654; Lane 3: 100bp ladder
 +
<br>&#9654; Lane 4: blank
 +
<br>&#9654; Lane 5: esa: 2 bands
 +
<br>&#9654; Lane 6: 1kb ladder
 +
<br>&#9654; Lane 7:blank
 +
</span>
 +
 
 +
</div>
 +
 
 
<br><br>
 
<br><br>
 +
 +
 
</div>
 
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 
</td>
 
</td>
 
</tr>
 
</tr>
 +
 +
<! ------------------------------- new day ----------------------------->
 +
 +
 +
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; June 20
 +
<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">
 +
Freezing of HEK293
 +
<br> &#9654; P6
 +
<br> &#9654; cells from a 150 mm dish and 90mm dish
 +
</span>
 +
 +
</div>
 +
 +
<br><br>
 +
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
 +
 +
 +
 +
<! ------------------------------- new day ----------------------------->
 +
  
  
Line 243: Line 516:
  
 
<! ------------------------------- Footer START ------------------------------------------------>
 
<! ------------------------------- Footer START ------------------------------------------------>
 +
  
 
<div id="footer1">
 
<div id="footer1">
Line 248: 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