Difference between revisions of "Team:SPSingapore/Notebook-Week-8"

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<li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook"><span>NOTEBOOK</span></a>
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  <ul><li class = 'last'>&nbsp;</li></ul></li>
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  <li><a href = "https://2015.igem.org/Team:SPSingapore/Protocol"><span>PROTOCOL</span></a>
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  <ul><li class = 'last'>&nbsp;</li></ul></li>
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   <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>
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<div style = "border-top: 5px solid blue; width:750px; text-align:justify;margin-bottom: 20px; line-height:normal;">
−
<h3 style ="margin-left:20px;">Week 8 (5/7 - 11/7)</h3>
+
<h3 style ="margin-left:20px;">Week 8 (12/7 - 18/7)</h3>
  
  
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<!--------------------new day------------------------------>
 +
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; July 13
 +
<br><br>
 +
<span class = "brown"> Anaerobic Promoter</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han & Yun Ting &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
Ligation for FNRgfp (4X)
 +
<br> 200ng of gfp plasmid (4.36ul)
 +
<br> 297.5 ng insert (7:1) 10.3
 +
<br> 1ul 10Buffer (53.34)
 +
<br> 4ul T4 ligase
 +
</span>
 +
<br><br>
 +
<span class = "nbcontent">
 +
Trial run of anaerobe chamber
 +
<br> open sachet to decrease O2 at 3.30pm
 +
<br> takes 2.5h to activate
 +
<br> streak plate of P. putida a strict aerobe and E. coli, facultative aerobe in chamber at 30deg C to grow O/N
 +
<br> P. putida is an obligate aerobe and if chamber works ,it will not grow
 +
<br> E. coli should grow in both conditions
 +
<br> controls grown outside chamber
 +
</span>
 +
 +
 +
 +
</div>
 +
 +
<br><br>
 +
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
  
 
<!----------------------new day------------------------------>
 
<!----------------------new day------------------------------>
  
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; July 14
 +
<br><br>
 +
<span class = "brown"> Anaerobic Promoter</span>
 +
&emsp;&emsp;<span class = "blue"> Invasin + Listerolysin</span>
 +
<br>
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
No colonies for FNRgfp plasmid
 +
</span>
 +
<br>
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td colspan = 2><b>Inv Plasmid Verification</b></td></tr>
 +
<tr><td> buffer </td><td> 1 </td></tr>
 +
<tr><td> EcoRI </td><td> 0.5 </td></tr>
 +
<tr><td> in plasmid </td><td> 0.5 </td></tr>
 +
<tr><td> dH2O </td><td> 8 </td></tr>
 +
<tr><td> Total </td><td> 10ul </td></tr>
 +
</table>
 +
<br><br>
  
 +
<span class = "nbcontent">
 +
Gel extraction RE digest of EsaR-GFP plasmid</span><br>
 +
<table class = "nbtable" style = "margin-left:50px">
 +
<tr><td></td><td><b>Ligation</b></td><td><b>Control</b></td></tr>
 +
<tr><td> Vector (45ng) </td><td> 1 </td><td> 1 </td></tr>
 +
<tr><td> insert </td><td> 1 </td><td> 1 </td></tr>
 +
<tr><td> Buffer </td><td> 1 </td><td> 1 </td></tr>
 +
<tr><td> dH2O </td><td> 6 </td><td> 7 </td></tr>
 +
<tr><td> ligase </td><td> 1 </td><td> 1 </td></tr>
 +
 +
</table>
 +
 +
<br><br>
 +
 +
<span class = "nbcontent">
 +
Transformation of FNRgfp
 +
<br> Anaerobe jar
 +
<br> E. coli grew in both conditions
 +
<br> P putida-> some growth in anaerobe chamber
 +
<br> Proper streak plate in aerobic conditions
 +
<br> Packet runs out after 16 hours
 +
</span>
 +
 +
</div>
 +
 +
<br><br>
 +
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
  
  
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 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; July 15
 +
<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">
 +
FNRgfp-> no colonies after transformation
 +
</span>
 +
<br>
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> religation (4X reaction) </td></tr>
 +
<tr><td> 200ng of gfp plasmid (4.4ul) </td></tr>
 +
<tr><td> 2125ng of insert (8ul) </td></tr>
 +
<tr><td> 4ul T4 ligase </td></tr>
 +
<tr><td> 55.6 H2O </td></tr>
 +
</table>
 +
 +
</div>
 +
 +
<br><br>
 +
 +
<span class = "brown"> Anaerobic Promoter</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han & Chi Yan &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
<span class = "nbcontent">
 +
Transformation of FNR gfp into 20ul of BL21
 +
</span>
 +
 +
</div>
 +
 +
<br><br>
 +
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
  
 
<!----------------------new day------------------------------>
 
<!----------------------new day------------------------------>
  
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; July 16
 +
<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">
 +
No colonies grew</span><br>
 +
<span class = "nbcontent">
 +
Ran a gel, FNRgfp pcr, gfp pcr, fnr, 100bp ladder
 +
<br> &#9654; Seems to be band shift.
 +
</span>
 +
</div>
  
 +
<br><br>
 +
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
  
  

Revision as of 18:28, 17 September 2015


Research Notebook

Week 8 (12/7 - 18/7)

▪ July 13

Anaerobic Promoter    ☀ Yi Han & Yun Ting ☀

Ligation for FNRgfp (4X)
200ng of gfp plasmid (4.36ul)
297.5 ng insert (7:1) 10.3
1ul 10Buffer (53.34)
4ul T4 ligase

Trial run of anaerobe chamber
open sachet to decrease O2 at 3.30pm
takes 2.5h to activate
streak plate of P. putida a strict aerobe and E. coli, facultative aerobe in chamber at 30deg C to grow O/N
P. putida is an obligate aerobe and if chamber works ,it will not grow
E. coli should grow in both conditions
controls grown outside chamber


▪ July 14

Anaerobic Promoter    Invasin + Listerolysin
☀ Yi Han ☀

No colonies for FNRgfp plasmid
Inv Plasmid Verification
buffer 1
EcoRI 0.5
in plasmid 0.5
dH2O 8
Total 10ul


Gel extraction RE digest of EsaR-GFP plasmid
LigationControl
Vector (45ng) 1 1
insert 1 1
Buffer 1 1
dH2O 6 7
ligase 1 1


Transformation of FNRgfp
Anaerobe jar
E. coli grew in both conditions
P putida-> some growth in anaerobe chamber
Proper streak plate in aerobic conditions
Packet runs out after 16 hours


▪ July 15

Anaerobic Promoter    ☀ Yi Han ☀

FNRgfp-> no colonies after transformation
religation (4X reaction)
200ng of gfp plasmid (4.4ul)
2125ng of insert (8ul)
4ul T4 ligase
55.6 H2O


Anaerobic Promoter    ☀ Yi Han & Chi Yan ☀

Transformation of FNR gfp into 20ul of BL21


▪ July 16

Anaerobic Promoter    ☀ Yi Han ☀

No colonies grew
Ran a gel, FNRgfp pcr, gfp pcr, fnr, 100bp ladder
▶ Seems to be band shift.