Difference between revisions of "Team:CGU Taiwan/Notebook"

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<section id="blog-details" class="padding-top">
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        <div class="container">
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            <div class="row">
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                    <div class="row" >
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                        <div class="col-md-12 col-sm-12"  >
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<table class="protocol-table" style="margin:auto">
 +
 +
<tr><td><a>Description</a></td><td><a>Yeast With IL-8 Receptor</a></td><td><a>Toehold Switch As RNA Senor</a></td><td><a>Prototype Design</a></td></tr>
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</table>
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</div>
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</div>
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</div>
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</div>
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</section>
  
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<div class="single-blog blog-details two-column" id="Protocols">
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<h2 class="post-title bold">GPCR</h2>
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</div>
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<div id="accordion-container">
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<div class="panel-group" id="accordion">
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 +
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<div class="panel panel-default">
 +
<div class="panel-heading">
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<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
 +
</h4>
 +
</div>
 +
<div id="collapse1" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
 +
Goal:
 +
<br>
 +
&nbsp;&nbsp;1. Extraction of gDNA of Far1∆::KANMX strain<br>
 +
&nbsp;&nbsp;2. Detection the concentration of fast extracted gDNA of ∆Far1 strain<br>
 +
&nbsp;&nbsp;3. PCR gDNA of Far1∆ ::KANMX<br>
 +
&nbsp;&nbsp;4. Electrophoresis to check PCR product<br>
 +
<br>
 +
Experiment steps:<br>
 +
< Extraction of gDNA of Far1∆::KANMX strain><br>
 +
Consult the protocol<protocol of fast extraction of gDNA of yeast><br>
  
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<Detection the concentration of fast extracted gDNA>
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<table class="protocol-table">
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<tr><th></th><th></th><th></th><th></th></tr>
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</thead>
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+
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 +
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
 +
</table>
 +
<br>
 +
<PCR gDNA of Far1∆::KANMX ><br>
 +
1. Design of primers<br>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> Name</td><td>Pur. </td><td>Seq.(5’-3’) </td><td>Size (mer.) </td><td> MW (g/mol)</td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
 +
<tr><td>dFAR1 F’</td><td>Desalt </td><td>ggTTTTgTTAggCgggCAAg </td><td>20 </td><td>6244.1 </td><td>53.8 </td><td>55</td><td>21.25</td><td>212.50</td></tr>
 +
<tr><td>dFAR1 R’</td><td>Desalt </td><td>CATTAACTgCTATTTACgACgC </td><td>22 </td><td>6669.4 </td><td>51.1 </td><td>41</td><td>17.12</td><td>171.20</td></tr>
 +
</table>
 +
<br>
 +
2. PCR program
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>Step </td><td>Temperature </td><td>Time</td></tr>
 +
<tr><td>Step1 </td><td>95℃ </td><td>5min</td></tr>
 +
<tr><td>Step2 </td><td>95℃ </td><td>30s</td></tr>
 +
<tr><td>Step3 </td><td>52℃ </td><td>30s</td></tr>
 +
<tr><td>Step4→step2 for 30 cycle </td><td>72℃ </td><td>2min </td></tr>
 +
<tr><td>Step5 </td><td>72℃ </td><td>5min </td></tr>
 +
<tr><td>Step6 (hold on) </td><td>10℃ </td><td>1hr </td></tr>
 +
 +
</table>
 +
<br>
 +
3.PCR reagent
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>10x Dream Taq buffer </td><td>2.5μl</td></tr>
 +
<tr><td>2.5mM dNTP</td><td>0.5μl</td></tr>
 +
<tr><td>10mM primer(F)</td><td>0.5μl</td></tr>
 +
<tr><td>10mM primer(R)</td><td>0.5μl </td></tr>
 +
<tr><td>template (Far1∆::KANMX strain gDNA) </td><td>3.4μl</td></tr>
 +
<tr><td>Taq polymerase</td><td>0.5μl</td></tr>
 +
<tr><td>ddH2O </td><td>17.1μl</td></tr>
 +
<tr><td>Total volume</td><td>25μl</td></tr>
 +
</table><br>
 +
<br>
 +
< Electrophoresis to check PCR product><br>
 +
Material: <br>
 +
&nbsp;&nbsp;DNA marker: 100bp ladder 8μl<br>
 +
&nbsp;&nbsp;DNA sample: 2μlPCR product + 1μl 6x loading buffer<br>
 +
Condition: <br>
 +
&nbsp;&nbsp;0.5xTBE buffer 100V<br>
 +
Time:<br>
 +
&nbsp;&nbsp;30min<br>
 +
Result:<br>
 +
<!--照片-->
 +
 +
&nbsp;&nbsp;M：Marker；#1：Far1 ∆ for annealing at 52℃<br>
 +
&nbsp;&nbsp;There is no band appears in the gel electrophoresis.<br>
 +
Conclusion:<br>
 +
&nbsp;&nbsp;Due to the Figure 1 in result, we have to check the temperature of primers annealing and the design of primers to solve the problem. Next, we use gradient PCR to find out the exactly temperature of primer annealing and we add positive control as well.
 +
 +
 +
</div>
 +
</div>
 +
</div>
 +
 +
<div class="panel panel-default">
 +
<div class="panel-heading">
 +
<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse2">2015.7.2</a>
 +
</h4>
 +
</div>
 +
<div id="collapse2" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
 +
Goal: <br>
 +
&nbsp;&nbsp;1. Extraction of gDNA of FAR1∆::KANMX strain <br>
 +
&nbsp;&nbsp;2. Detection the concentration of fast extracted gDNA of FAR1∆::KANMX strain<br>
 +
&nbsp;&nbsp;3. First round of PCR <br>
 +
&nbsp;&nbsp;4. Electrophoresis to check first round-PCR product<br>
 +
&nbsp;&nbsp;5. Second round of PCR <br>
 +
<br>
 +
Experiment steps:<br>
 +
< Extraction of gDNA of FAR1∆::KANMX strain><br>
 +
Consult the protocol <protocol of fast extraction of gDNA of yeast><br>
  
−
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display:inline;
 
−
 
−
 
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−
 
  
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<Detection the concentration of fast extracted gDNA>
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</style>
+
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 +
<tr><td>Far1∆::KANMX </td><td>1.72 </td><td>0.78 </td><td>42.7 </td></tr>
 +
<tr><td>Positive control </td><td>1.63 </td><td>0.76 </td><td>37.1 </td></tr>
 +
</table>
 +
 +
<First round of PCR ><br>
 +
1. Information of primers<br>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> Name</td><td>Pur. </td><td>Seq.(5’-3’) </td><td>Size (mer.) </td><td> MW (g/mol)</td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
 +
<tr><td>dFAR1 F’</td><td>Desalt </td><td>ggTTTTgTTAggCgggCAAg </td><td>20 </td><td>6244.1 </td><td>53.8 </td><td>55</td><td>21.25</td><td>212.50</td></tr>
 +
<tr><td>dFAR1 R’</td><td>Desalt </td><td>CATTAACTgCTATTTACgACgC </td><td>22 </td><td>6669.4 </td><td>51.1 </td><td>41</td><td>17.12</td><td>171.20</td></tr>
 +
</table>
 +
<br>
 +
2.PCR program<br>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>Step </td><td>Temperature </td><td>Time</td></tr>
 +
<tr><td>Step1 </td><td>95℃ </td><td>5min</td></tr>
 +
<tr><td>Step2 </td><td>95℃ </td><td>30s</td></tr>
 +
<tr><td>Step3 </td><td>Gradient42℃-46℃-50℃ </td><td>30s</td></tr>
 +
<tr><td>Step4→step2 for 30 cycle </td><td>72℃ </td><td>2min </td></tr>
 +
<tr><td>Step5 </td><td>72℃ </td><td>5min </td></tr>
 +
<tr><td>Step6 (hold on) </td><td>10℃ </td><td>1hr </td></tr>
 +
</table>
 +
<br>
 +
3.PCR reagent<br>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>10x Dream Taq buffer </td><td>2.5μl</td></tr>
 +
<tr><td>2.5mM dNTP</td><td>0.5μl</td></tr>
 +
<tr><td>10mM primer(F)</td><td>0.5μl</td></tr>
 +
<tr><td>10mM primer(R)</td><td>0.5μl </td></tr>
 +
<tr><td>template (Far1∆::KANMX strain gDNA) </td><td>3.4μl</td></tr>
 +
<tr><td>Taq polymerase</td><td>0.5μl</td></tr>
 +
<tr><td>ddH2O </td><td>17.1μl</td></tr>
 +
<tr><td>Total volume</td><td>25μl</td></tr>
 +
</table><br>
 +
<br>
 +
< Electrophoresis to check first round-PCR product (25μl ul)><br>
 +
Material:<br>
 +
&nbsp;&nbsp;DNA marker: 100bp ladder 8μl<br>
 +
&nbsp;&nbsp;DNA sample: 2μl PCR product + 1μl 6x loading buffer<br>
 +
Condition: <br>
 +
&nbsp;&nbsp;0.5xTBE buffer 100V <br>
 +
Time: <br>
 +
&nbsp;&nbsp;30min<br>
 +
Result:<br>
 +
<!--照片-->
 +
M: Marker; #1: FAR1∆ annealing at 42 ℃; #2: FAR1∆ annealing at 46 ℃;<br>
 +
#3: FAR1∆ annealing at 50 ℃; #4: FAR1∆ annealing at 52 ℃(positive control)
  
 +
 +
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
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<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
</table>
 +
</div>
 +
</div>
 +
</div>
 +
 +
<div class="panel panel-default">
 +
<div class="panel-heading">
 +
<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
 +
</h4>
 +
</div>
 +
<div id="collapse1" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan yun, Jinting , Jinye
 +
Goal:
 +
1. Extraction of gDNA of Far1∆::KANMX strain
 +
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 +
3. PCR gDNA of Far1∆ ::KANMX
 +
4. Electrophoresis to check PCR product
 +
Experiment steps:
 +
< Extraction of gDNA of Far1∆::KANMX strain>
 +
Consult the protocol<protocol of fast extraction of gDNA of yeast>
  
 +
<Detection the concentration fo fast extracted gDNA>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 +
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
 +
</table>
 +
 +
<PCR gDNA of Far1∆::KANMX >
 +
1. Design of primers
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
 +
</table>
 +
 +
 +
 +
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
</table>
 +
 +
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
</table>
 +
</div>
 +
</div>
 +
</div>
 +
 +
<div class="panel panel-default">
 +
<div class="panel-heading">
 +
<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
 +
</h4>
 +
</div>
 +
<div id="collapse1" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan yun, Jinting , Jinye
 +
Goal:
 +
1. Extraction of gDNA of Far1∆::KANMX strain
 +
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 +
3. PCR gDNA of Far1∆ ::KANMX
 +
4. Electrophoresis to check PCR product
 +
Experiment steps:
 +
< Extraction of gDNA of Far1∆::KANMX strain>
 +
Consult the protocol<protocol of fast extraction of gDNA of yeast>
  
−
<body>
+
<Detection the concentration fo fast extracted gDNA>
−
+
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 +
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
 +
</table>
 +
 +
<PCR gDNA of Far1∆::KANMX >
 +
1. Design of primers
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
 +
</table>
 +
 +
 +
 +
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
<tr><td> </td><td> </td></tr>
 +
</table>
 +
 +
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
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<tr><td> </td><td> </td></tr>
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<tr><td> </td><td> </td></tr>
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</table>
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
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</div>
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<div id="collapse1" class="panel-collapse collapse ">
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<div class="panel-body">
 +
Operator: Wan yun, Jinting , Jinye
 +
Goal:
 +
1. Extraction of gDNA of Far1∆::KANMX strain
 +
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 +
3. PCR gDNA of Far1∆ ::KANMX
 +
4. Electrophoresis to check PCR product
 +
Experiment steps:
 +
< Extraction of gDNA of Far1∆::KANMX strain>
 +
Consult the protocol<protocol of fast extraction of gDNA of yeast>
  
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    <section id="recent-projects" class="recent-projects">
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<Detection the concentration fo fast extracted gDNA>
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                <h1 class="title text-center wow fadeInDown" data-wow-duration="500ms" data-wow-delay="300ms">Welcome to CGU_Taiwan's Wiki page</h1>
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<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
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                <h2 class="text-center padding-bottom wow fadeInDown" data-wow-duration="400ms" data-wow-delay="400ms">Oral cancer is a common cancer worldwide. One of the major problems of oral cancer is that early malignancy could only be detected by clinical oral examination from health care professionals, and there are no clinical tests at the molecular level. Recently, there are several Proteins and mRNAs reported as promising potential non-invasive biomarkers in saliva for oral cancer. Our team wants to establish simple, objective and quantifiable non-invasive detection system for those biomarkers.
+
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
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<br>Our system includes two aims :
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</table>
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<br>1) engineered yeasts that express a reporter activated by IL-8, which is one of the most statistically significant protein biomarkers in oral cancer saliva samples.
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<br>2) Synthetic toehold switch gene regulators as RNA sensors to detect specific mRNA biomarkers. These efforts will provide the groundwork for new molecular diagnoses in oral cancer.</h2>
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<PCR gDNA of Far1∆::KANMX >
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1. Design of primers
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
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<div class="home-box2">
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
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</h4>
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Operator: Wan yun, Jinting , Jinye
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<div class="home-slider3 portfolio-single">
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Goal:
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<div class="home-box1">
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1. Extraction of gDNA of Far1∆::KANMX strain
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<img src="https://static.igem.org/mediawiki/2015/2/2f/CGU-home-logo.jpg" >
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    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
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</div>
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3. PCR gDNA of Far1∆ ::KANMX
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<div class="home-box2">
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4. Electrophoresis to check PCR product
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<img src="https://static.igem.org/mediawiki/2015/5/51/CGU-home-HP-258x266.jpeg">
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Experiment steps:
−
</div>
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< Extraction of gDNA of Far1∆::KANMX strain>
−
</div>
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Consult the protocol<protocol of fast extraction of gDNA of yeast>
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    <!--/#recent-projects-->
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    <section id="clients">
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<Detection the concentration fo fast extracted gDNA>
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        <div class="container">
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<table class="protocol-table">
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<thead>
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                    <div class="clients text-center wow fadeInUp" data-wow-duration="500ms" data-wow-delay="300ms">
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</thead>
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                        <p><img src="images/home/clients.png" class="img-responsive" alt=""></p>
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<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
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                        <h1 class="title">~~~~~~~~~~~</h1>
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<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
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                        <p>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ <br>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ </p>
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</table>
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                    </div>
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                    <div class="clients-logo wow fadeIn" data-wow-duration="1000ms" data-wow-delay="600ms">
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<PCR gDNA of Far1∆::KANMX >
−
                        <div class="col-xs-3 col-sm-2">
+
1. Design of primers
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                            <a href="#"><img src="images/home/client1.png" class="img-responsive" alt=""></a>
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<table class="protocol-table">
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</thead>
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                        </div>
+
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
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                        <div class="col-xs-3 col-sm-2">
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
−
                            <a href="#"><img src="images/home/client3.png" class="img-responsive" alt=""></a>
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
−
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<div class="panel-heading">
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<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
 +
</h4>
 +
</div>
 +
<div id="collapse1" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan yun, Jinting , Jinye
 +
Goal:
 +
1. Extraction of gDNA of Far1∆::KANMX strain
 +
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 +
3. PCR gDNA of Far1∆ ::KANMX
 +
4. Electrophoresis to check PCR product
 +
Experiment steps:
 +
< Extraction of gDNA of Far1∆::KANMX strain>
 +
Consult the protocol<protocol of fast extraction of gDNA of yeast>
  
 +
<Detection the concentration fo fast extracted gDNA>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 +
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
 +
</table>
 +
 +
<PCR gDNA of Far1∆::KANMX >
 +
1. Design of primers
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
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<div class="panel-heading">
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<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
 +
</h4>
 +
</div>
 +
<div id="collapse1" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan yun, Jinting , Jinye
 +
Goal:
 +
1. Extraction of gDNA of Far1∆::KANMX strain
 +
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 +
3. PCR gDNA of Far1∆ ::KANMX
 +
4. Electrophoresis to check PCR product
 +
Experiment steps:
 +
< Extraction of gDNA of Far1∆::KANMX strain>
 +
Consult the protocol<protocol of fast extraction of gDNA of yeast>
  
−
   
+
<Detection the concentration fo fast extracted gDNA>
−
+
<table class="protocol-table">
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+
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 +
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
 +
</table>
 +
 +
<PCR gDNA of Far1∆::KANMX >
 +
1. Design of primers
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th></tr>
 +
</thead>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
 +
<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
 +
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse1">2015.7.1</a>
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Operator: Wan yun, Jinting , Jinye
 +
Goal:
 +
1. Extraction of gDNA of Far1∆::KANMX strain
 +
    2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
 +
3. PCR gDNA of Far1∆ ::KANMX
 +
4. Electrophoresis to check PCR product
 +
Experiment steps:
 +
< Extraction of gDNA of Far1∆::KANMX strain>
 +
Consult the protocol<protocol of fast extraction of gDNA of yeast>
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<Detection the concentration fo fast extracted gDNA>
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<table class="protocol-table">
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<thead>
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<tr><th></th><th></th></tr>
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</thead>
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<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 +
<tr><td>Far1∆::KANMX strain </td><td>1.62 </td><td>0.97 </td><td>44.7 </td></tr>
 +
</table>
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<PCR gDNA of Far1∆::KANMX >
 +
1. Design of primers
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<table class="protocol-table">
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<thead>
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<tr><th></th><th></th></tr>
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</thead>
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td>Tm (℃)</td><td>GC (%)</td><td>Nmole</td><td>μl for 100μM </td></tr>
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>55</td><td>21.25</td><td>212.50</td></tr>
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<tr><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td>41</td><td>17.12</td><td>171.20</td></tr>
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Revision as of 14:33, 17 September 2015

Home | CGU_Taiwan

Home | CGU_Taiwan

DescriptionYeast With IL-8 ReceptorToehold Switch As RNA SenorPrototype Design

GPCR

2015.7.1

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Extraction of gDNA of Far1∆::KANMX strain
  2. Detection the concentration of fast extracted gDNA of ∆Far1 strain
  3. PCR gDNA of Far1∆ ::KANMX
  4. Electrophoresis to check PCR product

Experiment steps:
< Extraction of gDNA of Far1∆::KANMX strain>
Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7


1. Design of primers
NamePur. Seq.(5’-3’) Size (mer.) MW (g/mol)Tm (℃)GC (%)Nmoleμl for 100μM
dFAR1 F’Desalt ggTTTTgTTAggCgggCAAg 20 6244.1 53.8 5521.25212.50
dFAR1 R’Desalt CATTAACTgCTATTTACgACgC 22 6669.4 51.1 4117.12171.20

2. PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 52℃ 30s
Step4→step2 for 30 cycle 72℃ 2min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

3.PCR reagent
10x Dream Taq buffer 2.5μl
2.5mM dNTP0.5μl
10mM primer(F)0.5μl
10mM primer(R)0.5μl
template (Far1∆::KANMX strain gDNA) 3.4μl
Taq polymerase0.5μl
ddH2O 17.1μl
Total volume25μl


< Electrophoresis to check PCR product>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample: 2μlPCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:
  M：Marker；#1：Far1 ∆ for annealing at 52℃
  There is no band appears in the gel electrophoresis.
Conclusion:
  Due to the Figure 1 in result, we have to check the temperature of primers annealing and the design of primers to solve the problem. Next, we use gradient PCR to find out the exactly temperature of primer annealing and we add positive control as well.

2015.7.2

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Extraction of gDNA of FAR1∆::KANMX strain
  2. Detection the concentration of fast extracted gDNA of FAR1∆::KANMX strain
  3. First round of PCR
  4. Electrophoresis to check first round-PCR product
  5. Second round of PCR

Experiment steps:
< Extraction of gDNA of FAR1∆::KANMX strain>
Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX 1.72 0.78 42.7
Positive control 1.63 0.76 37.1

1. Information of primers
NamePur. Seq.(5’-3’) Size (mer.) MW (g/mol)Tm (℃)GC (%)Nmoleμl for 100μM
dFAR1 F’Desalt ggTTTTgTTAggCgggCAAg 20 6244.1 53.8 5521.25212.50
dFAR1 R’Desalt CATTAACTgCTATTTACgACgC 22 6669.4 51.1 4117.12171.20

2.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 Gradient42℃-46℃-50℃ 30s
Step4→step2 for 30 cycle 72℃ 2min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

3.PCR reagent
10x Dream Taq buffer 2.5μl
2.5mM dNTP0.5μl
10mM primer(F)0.5μl
10mM primer(R)0.5μl
template (Far1∆::KANMX strain gDNA) 3.4μl
Taq polymerase0.5μl
ddH2O 17.1μl
Total volume25μl


< Electrophoresis to check first round-PCR product (25μl ul)>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample: 2μl PCR product + 1μl 6x loading buffer
Condition:
  0.5xTBE buffer 100V
Time:
  30min
Result:
M: Marker; #1: FAR1∆ annealing at 42 ℃; #2: FAR1∆ annealing at 46 ℃;
#3: FAR1∆ annealing at 50 ℃; #4: FAR1∆ annealing at 52 ℃(positive control)

2015.7.1

Operator: Wan yun, Jinting , Jinye Goal: 1. Extraction of gDNA of Far1∆::KANMX strain 2. Detection the concentration of fast extracted gDNA of ∆Far1 strain 3. PCR gDNA of Far1∆ ::KANMX 4. Electrophoresis to check PCR product Experiment steps: < Extraction of gDNA of Far1∆::KANMX strain> Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7
1. Design of primers
Tm (℃)GC (%)Nmoleμl for 100μM
5521.25212.50
4117.12171.20

2015.7.1

Operator: Wan yun, Jinting , Jinye Goal: 1. Extraction of gDNA of Far1∆::KANMX strain 2. Detection the concentration of fast extracted gDNA of ∆Far1 strain 3. PCR gDNA of Far1∆ ::KANMX 4. Electrophoresis to check PCR product Experiment steps: < Extraction of gDNA of Far1∆::KANMX strain> Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7
1. Design of primers
Tm (℃)GC (%)Nmoleμl for 100μM
5521.25212.50
4117.12171.20

2015.7.1

Operator: Wan yun, Jinting , Jinye Goal: 1. Extraction of gDNA of Far1∆::KANMX strain 2. Detection the concentration of fast extracted gDNA of ∆Far1 strain 3. PCR gDNA of Far1∆ ::KANMX 4. Electrophoresis to check PCR product Experiment steps: < Extraction of gDNA of Far1∆::KANMX strain> Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7
1. Design of primers
Tm (℃)GC (%)Nmoleμl for 100μM
5521.25212.50
4117.12171.20

2015.7.1

Operator: Wan yun, Jinting , Jinye Goal: 1. Extraction of gDNA of Far1∆::KANMX strain 2. Detection the concentration of fast extracted gDNA of ∆Far1 strain 3. PCR gDNA of Far1∆ ::KANMX 4. Electrophoresis to check PCR product Experiment steps: < Extraction of gDNA of Far1∆::KANMX strain> Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7
1. Design of primers
Tm (℃)GC (%)Nmoleμl for 100μM
5521.25212.50
4117.12171.20

2015.7.1

Operator: Wan yun, Jinting , Jinye Goal: 1. Extraction of gDNA of Far1∆::KANMX strain 2. Detection the concentration of fast extracted gDNA of ∆Far1 strain 3. PCR gDNA of Far1∆ ::KANMX 4. Electrophoresis to check PCR product Experiment steps: < Extraction of gDNA of Far1∆::KANMX strain> Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7
1. Design of primers
Tm (℃)GC (%)Nmoleμl for 100μM
5521.25212.50
4117.12171.20

2015.7.1

Operator: Wan yun, Jinting , Jinye Goal: 1. Extraction of gDNA of Far1∆::KANMX strain 2. Detection the concentration of fast extracted gDNA of ∆Far1 strain 3. PCR gDNA of Far1∆ ::KANMX 4. Electrophoresis to check PCR product Experiment steps: < Extraction of gDNA of Far1∆::KANMX strain> Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7
1. Design of primers
Tm (℃)GC (%)Nmoleμl for 100μM
5521.25212.50
4117.12171.20

RNA

2015.7.1

Operator: Wan yun, Jinting , Jinye Goal: 1. Extraction of gDNA of Far1∆::KANMX strain 2. Detection the concentration of fast extracted gDNA of ∆Far1 strain 3. PCR gDNA of Far1∆ ::KANMX 4. Electrophoresis to check PCR product Experiment steps: < Extraction of gDNA of Far1∆::KANMX strain> Consult the protocol
Strains 260/280 260/230 C(ng/μl)
Far1∆::KANMX strain 1.62 0.97 44.7
1. Design of primers
Tm (℃)GC (%)Nmoleμl for 100μM
5521.25212.50
4117.12171.20