Difference between revisions of "Team:SPSingapore/Notebook-Week-6"

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        <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>
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<li><a href = "https://2015.igem.org/Team:SPSingapore/Notebook"><span>NOTEBOOK</span></a>
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         <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>
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         <li class = 'last'><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-17"><span>Week 17 (13/8 - 17/9)</span></a></li>
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         <li class = 'last'><a href = "https://2015.igem.org/Team:SPSingapore/Notebook-Week-17"><span>Week 17 (13/9 - 18/9)</span></a></li>
 
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<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">
 +
&#9642; June 28
 +
<br><br>
 +
<span class = "black"> esa Quorum Sensing</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Adrian &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
 +
<span class = "nbcontent">
 +
BBPrefix_esaRBS PCR
 +
</span>
 +
<br>
 +
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> H2O </td><td> 221ul </td></tr>
 +
<tr><td> Buffer </td><td> 80ul </td></tr>
 +
<tr><td> MgCl1 </td><td> 32ul </td></tr>
 +
<tr><td> dNTP </td><td> 32ul </td></tr>
 +
<tr><td> FP_Bioricks Prefix </td><td> 16ul </td></tr>
 +
<tr><td> ORP_esaRBS_fragsyn </td><td> 16ul </td></tr>
 +
<tr><td> synpart_BBP_esaRBS </td><td> 1ul </td></tr>
 +
<tr><td> gotaq </td><td> 2ul </td></tr>
 +
<tr><td> Total </td><td> 400ul </td></tr>
 +
</table>
 +
<span>
 +
&#9654; Success-> PCR purification
 +
</span>
 +
<br><br>
 +
 +
<span class = "nbcontent">
 +
esaRBS_GFP_BBsuffix
 +
</span>
 +
<br>
 +
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> H2O </td><td> 237ul </td></tr>
 +
<tr><td> Buffer </td><td> 80ul </td></tr>
 +
<tr><td> MgCl2 </td><td> 32ul </td></tr>
 +
<tr><td> dNTP </td><td> 32ul </td></tr>
 +
<tr><td> RP_BB_suffix </td><td> 16ul </td></tr>
 +
<tr><td> ORP_esaRS_GFP </td><td> 16ul </td></tr>
 +
<tr><td> plac_GFPPLASMID </td><td> 1ul </td></tr>
 +
<tr><td> gotaq </td><td> 2ul </td></tr>
 +
</table>
 +
 +
<span>
 +
&#9654; Failed -> troubleshooting-> new OFP_esaRBBS_GFP
 +
<br> &emsp;  &emsp; Redid 30/6 with new primers
 +
<br> &emsp;  &emsp; Success with new primers-> fusion pcr
 +
</span>
 +
 +
 +
</div>
 +
 +
<br><br>
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
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</td>
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</tr>
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 +
  
  
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 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; June 29
 +
<br><br>
 +
<span class = "black" style = "background:green"> Lab Safety</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; SPSingapore Team &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
 +
<span class = "nbcontent">
 +
Safety Inspection for our lab by OSHE. We passed with flying colours!
 +
</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 2
 +
<br><br>
 +
<span class = "black"> esa Quorum Sensing</span>
 +
&emsp;&emsp;
 +
<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Clarice &#9728;</font>
 +
<br><br>
 +
<div class = "divnbcontent">
 +
 +
<span class = "nbcontent">
 +
Fusion PCR with esaRBS, GFP
 +
</span>
 +
<br>
 +
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> H2O </td><td> 104.07ul </td></tr>
 +
<tr><td> Buffer </td><td> 40ul </td></tr>
 +
<tr><td> MgCl2 </td><td> 16ul </td></tr>
 +
<tr><td> dNTPs </td><td> 16ul </td></tr>
 +
<tr><td> FP_BB_prefix </td><td> 8ul </td></tr>
 +
<tr><td> RP_BB_prefix </td><td> 8ul </td></tr>
 +
<tr><td> 400ng esaRBS </td><td> 2.13ul </td></tr>
 +
<tr><td> 400ng esaRBSgfp </td><td> 4.8ul </td></tr>
 +
<tr><td> gotaq </td><td> 1ul </td></tr>
 +
<tr><td> total </td><td> 200ul </td></tr>
 +
</table>
 +
<br>
 +
<span>
 +
&#9654; annealing temp 50degC
 +
<br> &#9654; Gel electrophoresis: looks correct-> PCR Purification
 +
</span>
 +
<br><br>
 +
 +
<span class = "nbcontent">
 +
RE digest of esaRBS+GFP insert
 +
</span>
 +
<br>
 +
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> Buffer </td><td> 2 </td></tr>
 +
<tr><td> EcoRI/PstI </td><td> 1/1 </td></tr>
 +
<tr><td> Template </td><td> 2ug 5ul </td></tr>
 +
<tr><td> dH2O </td><td> 11ul </td></tr>
 +
<tr><td> Total </td><td> 20ul </td></tr>
 +
</table>
 +
<br><br>
 +
 +
 +
<span class = "nbcontent">
 +
RE digest of esaRBS+GFP  vector
 +
</span>
 +
<br>
 +
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> Buffer </td><td> 2ul </td></tr>
 +
<tr><td> EcoRI/PstI </td><td> 1/1 </td></tr>
 +
<tr><td> Template 4ul </td><td> 2ug 5ul </td></tr>
 +
<tr><td> dH2O </td><td> 12ul </td></tr>
 +
<tr><td> Total </td><td> 20ul </td></tr>
 +
 +
</table>
 +
 +
 +
 +
 +
</div>
 +
 +
<br><br>
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
 +
 +
 +
<!--------------------new day ------------------------------------->
 +
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; July 3
 +
<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">
 +
Ran gel of gfp plasmid EcoRI/PstI, took out gel slice for vector - 4kb
 +
<br> &#9654; gel extraction
 +
<br> &#9654; gfpvector->38.7ng/ul
 +
<br> &#9654; fragment-> direction purification 226.4ng/ul
 +
</span><br><br>
 +
<span class = "nbcontent">
 +
Ligation reaction (5:1) 6X
 +
</span>
 +
<table class = "nbtable" style = "margin-left:50px;">
 +
<tr><td> 300ng vector 7.8ng/ul </td></tr>
 +
<tr><td> 531.2ng of insert 2.4ul </td></tr>
 +
<tr><td> 12ul buffer </td></tr>
 +
<tr><td> 6ul enz </td></tr>
 +
<tr><td> 91.8ul H2O </td></tr>
 +
</table>
 +
<br>
 +
 +
<span>
 +
&#9654; ligate for 2 hours at 16 hours
 +
<br> &#9654; transform, plate, grow ON
 +
</span>
 +
 +
</div>
 +
 +
<br><br>
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
 +
 +
<!----------------------------new day ---------------------------->
 +
 +
 +
<tr class = "tablenotebook"><td><div class = "paper">
 +
&#9642; July 4
 +
<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-> ligation did not work
 +
</span><br><br>
 +
<span class = "nbcontent">
 +
Recalculate ligation reaction (3:1),7x
 +
</span>
 +
<table class = "nbtable" style = "margin-left: 50px;">
 +
<tr><td> 350ng vector->9ul vector </td></tr>
 +
<tr><td> 446.3ng insert-> 2ul </td></tr>
 +
<tr><td> 7ul ligase </td></tr>
 +
<tr><td> 110 H2O </td></tr>
 +
<tr><td> 12ul buffer </td></tr>
 +
</table>
 +
<br>
 +
 +
<span>
 +
&#9654; ligate for 2 hours at 16 hours
 +
<br> &#9654; transform, plate, grow ON
 +
</span>
 +
 +
</div>
 +
 +
<br><br>
 +
 +
</div>
 +
<div style = "border-top: 2px solid turquoise;"></div>
 +
</td>
 +
</tr>
 +
 +
 +
 +
<!----------------------------new day--------------------------------->
 
</table>
 
</table>
 
</center>
 
</center>
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<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;">
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<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;">
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<img src = "https://static.igem.org/mediawiki/2015/7/72/SPSingapore_SPS-Logo.png" height = "60px" style = "margin-top:10px;margin-right:50px;"></a>
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Latest revision as of 23:17, 18 September 2015


Research Notebook

Week 6 (28/6 - 4/7)

▪ June 28

esa Quorum Sensing    ☀ Adrian ☀

BBPrefix_esaRBS PCR
H2O 221ul
Buffer 80ul
MgCl1 32ul
dNTP 32ul
FP_Bioricks Prefix 16ul
ORP_esaRBS_fragsyn 16ul
synpart_BBP_esaRBS 1ul
gotaq 2ul
Total 400ul
▶ Success-> PCR purification

esaRBS_GFP_BBsuffix
H2O 237ul
Buffer 80ul
MgCl2 32ul
dNTP 32ul
RP_BB_suffix 16ul
ORP_esaRS_GFP 16ul
plac_GFPPLASMID 1ul
gotaq 2ul
▶ Failed -> troubleshooting-> new OFP_esaRBBS_GFP
    Redid 30/6 with new primers
    Success with new primers-> fusion pcr


▪ June 29

Lab Safety    ☀ SPSingapore Team ☀

Safety Inspection for our lab by OSHE. We passed with flying colours!


▪ July 2

esa Quorum Sensing    ☀ Clarice ☀

Fusion PCR with esaRBS, GFP
H2O 104.07ul
Buffer 40ul
MgCl2 16ul
dNTPs 16ul
FP_BB_prefix 8ul
RP_BB_prefix 8ul
400ng esaRBS 2.13ul
400ng esaRBSgfp 4.8ul
gotaq 1ul
total 200ul

▶ annealing temp 50degC
▶ Gel electrophoresis: looks correct-> PCR Purification

RE digest of esaRBS+GFP insert
Buffer 2
EcoRI/PstI 1/1
Template 2ug 5ul
dH2O 11ul
Total 20ul


RE digest of esaRBS+GFP vector
Buffer 2ul
EcoRI/PstI 1/1
Template 4ul 2ug 5ul
dH2O 12ul
Total 20ul


▪ July 3

Anaerobic Promoter    ☀ Yi Han ☀

Ran gel of gfp plasmid EcoRI/PstI, took out gel slice for vector - 4kb
▶ gel extraction
▶ gfpvector->38.7ng/ul
▶ fragment-> direction purification 226.4ng/ul

Ligation reaction (5:1) 6X
300ng vector 7.8ng/ul
531.2ng of insert 2.4ul
12ul buffer
6ul enz
91.8ul H2O

▶ ligate for 2 hours at 16 hours
▶ transform, plate, grow ON


▪ July 4

Anaerobic Promoter    ☀ Yi Han ☀

No colonies-> ligation did not work

Recalculate ligation reaction (3:1),7x
350ng vector->9ul vector
446.3ng insert-> 2ul
7ul ligase
110 H2O
12ul buffer

▶ ligate for 2 hours at 16 hours
▶ transform, plate, grow ON