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

Line 79: Line 79:
 
&nbsp;&nbsp;3. PCR gDNA of Far1∆ ::KANMX<br>
 
&nbsp;&nbsp;3. PCR gDNA of Far1∆ ::KANMX<br>
 
&nbsp;&nbsp;4. Electrophoresis to check PCR product<br>
 
&nbsp;&nbsp;4. Electrophoresis to check PCR product<br>
 +
<br>
 
Experiment steps:<br>
 
Experiment steps:<br>
 
< Extraction of gDNA of Far1∆::KANMX strain><br>
 
< Extraction of gDNA of Far1∆::KANMX strain><br>
Line 102: Line 103:
 
<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>
 
<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>
 
</table>
−
+
<br>
 
2. PCR program
 
2. PCR program
 
<table class="protocol-table">
 
<table class="protocol-table">
Line 117: Line 118:
 
 
 
</table>
 
</table>
−
+
<br>
 
3.PCR reagent
 
3.PCR reagent
 
<table class="protocol-table">
 
<table class="protocol-table">
Line 135: Line 136:
 
< Electrophoresis to check PCR product><br>
 
< Electrophoresis to check PCR product><br>
 
Material: <br>
 
Material: <br>
−
DNA marker: 100bp ladder 8μl<br>
+
&nbsp;&nbsp;DNA marker: 100bp ladder 8μl<br>
−
DNA sample: 2μlPCR product + 1μl 6x loading buffer<br>
+
&nbsp;&nbsp;DNA sample: 2μlPCR product + 1μl 6x loading buffer<br>
 
Condition: <br>
 
Condition: <br>
−
0.5xTBE buffer 100V<br>
+
&nbsp;&nbsp;0.5xTBE buffer 100V<br>
−
Time:30min<br>
+
Time:<br>
 +
&nbsp;&nbsp;30min<br>
 
Result:<br>
 
Result:<br>
 
<!--照片-->
 
<!--照片-->
 
 
−
M：Marker；#1：Far1 ∆ for annealing at 52℃
+
M：Marker；#1：Far1 ∆ for annealing at 52℃<br>
−
There is no band appears in the gel electrophoresis.
+
There is no band appears in the gel electrophoresis.<br>
−
Conclusion:
+
Conclusion:<br>
 
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.
 
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.
 
 
Line 168: Line 170:
 
&nbsp;&nbsp;4. Electrophoresis to check first round-PCR product<br>
 
&nbsp;&nbsp;4. Electrophoresis to check first round-PCR product<br>
 
&nbsp;&nbsp;5. Second round of PCR <br>
 
&nbsp;&nbsp;5. Second round of PCR <br>
 +
<br>
 
Experiment steps:<br>
 
Experiment steps:<br>
 
< Extraction of gDNA of FAR1∆::KANMX strain><br>
 
< Extraction of gDNA of FAR1∆::KANMX strain><br>
Line 176: Line 179:
 
<table class="protocol-table">
 
<table class="protocol-table">
 
<thead>
 
<thead>
−
<tr><th></th><th></th></tr>
+
<tr><th></th><th></th><th></th><th></th></tr>
 
</thead>
 
</thead>
 
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
 
<tr><td>Strains </td><td>260/280 </td><td>260/230 </td><td>C(ng/μl) </td></tr>
Line 193: Line 196:
 
<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>
 
<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>
 
</table>
 +
<br>
 
2.PCR program<br>
 
2.PCR program<br>
 
<table class="protocol-table">
 
<table class="protocol-table">
Line 206: Line 210:
 
<tr><td>Step6 (hold on) </td><td>10℃ </td><td>1hr </td></tr>
 
<tr><td>Step6 (hold on) </td><td>10℃ </td><td>1hr </td></tr>
 
</table>
 
</table>
 +
<br>
 
3.PCR reagent<br>
 
3.PCR reagent<br>
 
<table class="protocol-table">
 
<table class="protocol-table">
Line 226: Line 231:
 
Condition: <br>
 
Condition: <br>
 
&nbsp;&nbsp;0.5xTBE buffer 100V <br>
 
&nbsp;&nbsp;0.5xTBE buffer 100V <br>
−
Time:  
+
Time: <br>
 
&nbsp;&nbsp;30min<br>
 
&nbsp;&nbsp;30min<br>
 
Result:<br>
 
Result:<br>
 
<!--照片-->
 
<!--照片-->
−
M: Marker; #1: FAR1∆ annealing at 42 ℃; #2: FAR1∆ annealing at 46 ℃; #3: FAR1∆ annealing at 50 ℃; #4: FAR1∆ annealing at 52 ℃(positive control)
+
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)
  
 
 

Revision as of 13:14, 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