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

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</div>
 
</div>
 
 
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<div class="panel panel-default">
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<div class="panel-heading">
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<h4 class="panel-title">
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse24">2015.7.24</a>
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</h4>
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</div>
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<div id="collapse24" class="panel-collapse collapse ">
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<div class="panel-body">
 +
Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
 +
Goal: <br>
 +
&nbsp;&nbsp;1. Ligation of the rho-CXCR1 and p426 Gal<br>
 +
&nbsp;&nbsp;2. Transform the ligation product into the E.coli<br>
 +
Experimental steps:<br>
 +
< Ligation of the rho-CXCR1 and p426 Gal><br>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th>  </th><th>  </th><th>  </th></tr>
 +
</thead>
 +
<tr><td>&nbsp; </td><td>1:3 </td><td>Vector only </td></tr>
 +
<tr><td>Vector(11.1 ng/μl) </td><td>10μl </td><td>10μl </td></tr>
 +
<tr><td>Insert(9.1 ng/μl) </td><td>7μl </td><td>0μl </td></tr>
 +
<tr><td>Ligase </td><td>1μl </td><td>1μl </td></tr>
 +
<tr><td>Ligation buff. </td><td>2μl </td><td>2μl </td></tr>
 +
<tr><td>ddH2O </td><td>0μl </td><td>7μl </td></tr>
 +
<tr><td>total </td><td>20μl </td><td>20μl </td></tr>
 +
</table>
 +
Incubate the ligation product at R.T. for 2 hr.<br>
 +
<br>
 +
< Transform the ligation product into the competent cell><br>
 +
Consult the protocol < protocol of ligation and transformation of E.coli><br>
 +
</div>
 +
</div>
 +
</div>
 
 
 +
<div class="panel panel-default">
 +
<div class="panel-heading">
 +
<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse27">2015.7.27</a>
 +
</h4>
 +
</div>
 +
<div id="collapse27" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan-Yun, Jin-Ting<br>
 +
Goal: <br>
 +
&nbsp;&nbsp;1. Fast extraction of gDNA of Far1Δ::KanMX-FUS1-GFP<br>
 +
&nbsp;&nbsp;2. Check PCR for far1Δ::KanMX-FUS-GFP<br>
 +
Experimental steps:
 +
< Fast extraction of gDNA of far1Δ::KanMX-FUS-GFP >
 +
Consult the protocol <protocol of fast extraction of gDNA of yeast>
 +
<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>1.85 </td><td>1.19 </td><td>177.3 </td></tr>
 +
<tr><td>FAR1∆::KANMX  </td><td>1.83 </td><td>1.19 </td><td>163.8 </td></tr>
 +
<tr><td>Positive control  </td><td>1.96 </td><td>1.61 </td><td>235.3 </td></tr>
 +
</table>
 +
<br>
 +
< Check PCR for Far1Δ::KanMX-FUS1-GFP><br>
 +
1.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>46℃</td><td>30s</td></tr>
 +
<tr><td>Step4→step2 for 30 cycle </td><td>72℃ </td><td>90sec </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>
 +
<br>
 +
2.PCR reagent<br>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th></th><th></th><th></th></tr>
 +
</thead>
 +
<tr><td>&nbsp; </td><td>FAR1∆::KANMX  </td><td>Positive control  </td></tr>
 +
<tr><td>10x Dream Taq buffer </td><td>2.5μl</td><td>2.5μl</td></tr>
 +
<tr><td>2.5mM dNTP</td><td>0.5μl</td><td>0.5μl</td></tr>
 +
<tr><td>10mM primer(F)</td><td>0.5μl</td><td>0.5μl</td></tr>
 +
<tr><td>10mM primer(R)</td><td>0.5μl </td><td>0.5μl </td></tr>
 +
<tr><td>template (First round PCR product) </td><td>0.92μl</td><td>0.64μl</td></tr>
 +
<tr><td>Taq polymerase</td><td>0.5μl</td><td>0.5μl</td></tr>
 +
<tr><td>ddH2O </td><td>19.58μl</td><td>19.86μl</td></tr>
 +
<tr><td>Total volume</td><td>25μl</td><td>25μl</td></tr>
 +
</table><br>
 +
</div>
 +
</div>
 +
</div>
 +
 
 
 +
<div class="panel panel-default">
 +
<div class="panel-heading">
 +
<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse28">2015.7.28</a>
 +
</h4>
 +
</div>
 +
<div id="collapse28" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
 +
Goal: <br>
 +
&nbsp;&nbsp;1.Fast extraction of pGAL426 rho-CXCR1<br>
 +
Experimental steps: <br>
 +
< Fast extraction of plasmid DNA ><br>
 +
&nbsp;&nbsp;1. Add 50 μl of STE buffer(100 mM NaCl, 10 mM Tris buffer, pH 7.0, 1 mM EDTA) into each new Eppendorf tube.<br>
 +
&nbsp;&nbsp;2. Add 50 μl of Phenol chloroform and vortex vigorously for 30 sec.<br>
 +
&nbsp;&nbsp;3. Centrifuge at 13,000 g for 5 min.<br>
 +
&nbsp;&nbsp;4. Remove the 10 μl of supernatant to a new Eppendorf tube and run the sample on the 1 % agarose gel.<br>
 +
</div>
 +
</div>
 +
</div>
 
 
 +
 +
 
 
−
+
<div class="panel panel-default">
−
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<div class="panel-heading">
−
+
<h4 class="panel-title">
−
+
<a data-toggle="collapse" data-parent="#accordion" href="#collapse29">2015.7.29</a>
−
+
</h4>
 +
</div>
 +
<div id="collapse29" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan-Yun, Jin-Ting<br>
 +
Goal: <br>
 +
&nbsp;&nbsp;1. Miniprep of p426 Gal-rho-CXCR1<br>
 +
&nbsp;&nbsp;2. Enzyme digestion to check p426 Gal-rho-CXCR1 <br>
 +
Experimental steps: <br>
 +
< Miniprep of p426 Gal-rho-CXCR1><br>
 +
Consult the protocol <protocol of miniprep plamid><br>
 +
<br>
 +
< Enzyme digestion for the p426 Gal-rho-CXCR1 check ><br>
 +
1. Enzyme digestion<br>
 +
<table class="protocol-table">
 +
<thead>
 +
<tr><th>  </th><th>  </th><th>  </th></tr>
 +
</thead>
 +
<tr><td>p426 Gal-rho-CXCR1 </td><td>#7,9 </td><td>+,#8,12,16 </td></tr>
 +
<tr><td>10x NEB buff.4 </td><td>2.5μl </td><td>2.5μl </td></tr>
 +
<tr><td>Eco RI </td><td>0.5μl </td><td>0.5μl </td></tr>
 +
<tr><td>Bam HI </td><td>0.5μl </td><td>0.5μl </td></tr>
 +
<tr><td>DNA </td><td>2μl </td><td>1μl </td></tr>
 +
<tr><td>ddH2O </td><td>19.5μl </td><td>20.5μl </td></tr>
 +
<tr><td>total </td><td>25μl </td><td>25μl </td></tr>
 +
</table><br>
 +
<br>
 +
2. Loading 10 μl sample and 2 μl DNA loading dye into the 1 % agarose gel.
 +
<img src="" >
 +
 +
</div>
 +
</div>
 +
</div>
 +
 +
<div class="panel panel-default">
 +
<div class="panel-heading">
 +
<h4 class="panel-title">
 +
<a data-toggle="collapse" data-parent="#accordion" href="#collapse30">2015.7.30</a>
 +
</h4>
 +
</div>
 +
<div id="collapse30" class="panel-collapse collapse ">
 +
<div class="panel-body">
 +
Operator: Wan-Yun, Jin-Ting, Jin-Ye<br>
 +
Goal: <br>
 +
&nbsp;&nbsp;1. Transform pSB1C3 BBa_J04450 into the competent cell<br>
 +
&nbsp;&nbsp;2. Transform the plasmid (pRS405) into the competent cell<br>
 +
Experimental steps: <br>
 +
< Transform pSB1C3 BBa_J04450 into the competent cell><br>
 +
Consult the protocol <protocol of ligation and transformation of E.coli><br>
 +
Selection plate: LB+ Cam plate<br>
 +
<br>
 +
< Transform the plasmids (pRS405) into the competent cell><br>
 +
Selection plate: LB+ Amp plate<br>
 +
</div>
 +
</div>
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</div>
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<!--日期最小單位-->
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</div>
 
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<!--日期最小單位-->
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Revision as of 18:37, 18 September 2015

Home | CGU_Taiwan

Home | CGU_Taiwan

Yeast With IL-8 ReceptorToehold Switches as RNA sensor

Lab Note


Yeast With IL-8 Receptor

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)

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


2.PCR reagent
10x Dream Taq buffer 5μl
2.5mM dNTP1μl
10mM primer(F)1μl
10mM primer(R)1μl
template (First round PCR product) 1μl
Taq polymerase1μl
ddH2O 40μl
Total volume50μl

2015.7.3

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. 1.Electrophoresis to check second round-PCR product
  2. Transform PCR product into FUS::GFP(His) strain
Experiment steps:
< Electrophoresis to check second round-PCR product (50μl)>
Material:
  DNA marker: 100bp ladder 8μl   DNA sample:2μl PCR product + 1ul 6x loading buffer Condition: 0.5xTBE buffer 100V
Time: 30min

Result:
Figure 1. Gel electrophoresis of FAR1∆
M: Marker; #1: FAR1∆ annealing at 46 ℃

Consult the protocol < protocol of yeast transformation>
Use strain name: FUS1-GFP(His)
Selection plate: YPD+G418

2015.7.6

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Check transformation result
  2. Second round PCR
  3. Electrophoresis to check PCR product
  4. Incubate E.coli with shuttle vector-p426GAL1 from stock
Experiment steps:

1. Take plates out from incubator and observe growth of colony
2. Conclusion: We failed to transformation of PCR product so we should it again.


Consult the experiment record <2015.7.2 Experiment Record>

< Electrophoresis to check second round-PCR product>
Material:
  DNA marker: 100bp ladder 8μl
  DNA sample:2μlPCR product + 1μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result: Conclusion: Its expected length is 1.9kb and it worked.

< Incubate E.coli with shuttle vector-p426GAL1 from stock>
1. Take stock E.coli with shuttle vector-p426GAL1 from -80℃ fridge.
2. Plate E.coli on LB+Amp plates.
3. Incubate in 37℃ overnight.

2015.7.7

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Transform PCR product into FUS::GFP (His) strain
Experiment steps:

Consult the experiment record <2015.7.3 Experiment Record>

2015.7.9

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Miniprep plasmid of p426GAL1
  2. Measure concentration of plasmid.

Experiment steps:
< Miniprep plasmid of p426GAL1
Consult the protocol


  concentration 260/280 260/230
P426GAL1/1130.9ng/μl1.91 2.36
P426GAL1/2121.3ng/μl1.89 2.26

2015.7.12

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Second round PCR
  2. Incubate FAR1△::KANMX strain in 5ml YPD+A medium.
Experiment steps:

Consult the experiment record <2015.7.2 Experiment Record>

2015.7.17

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Transform PCR product into FUS1-GFP strain
Experiment steps:
< Transform PCR product into FUS1-GFP strain>
Consult the experiment record <2015.7.3 Experiment Record>

2015.7.20

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Maintain the colonies of far1Δ::KanMX-FUS1-GFP.
  2. 2nd round PCR for far1Δ::KanMX
  3. Phenol chloroform and EtOH precipitation of 2nd round PCR product
Experiment steps:
< Maintain the colonies of far1Δ::KanMX-FUS1-GFP >
  1. Choose 10 colonies to transfer the new YPD+G418 plate.
  2. Check plates after two days.

<2nd round PCR for far1Δ::KanMX
1.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 46℃30s
Step4→step2 for 30 cycle 72℃ 2min
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

2.PCR reagent
10x Dream Taq buffer 5μl
2.5mM dNTP1μl
10mM primer(F)1μl
10mM primer(R)1μl
template (First round PCR product) 1μl
Taq polymerase1μl
ddH2O 40μl
Total volume50μl


< Phenol chloroform and EtOH precipitation of 2nd round PCR product >
Consult the protocol

2015.7.21

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Transform PCR product(Far1Δ::KanMX) into FUS1-GFP strain
  2. Extraction of gDNA of yeast
  3. Check PCR for far1Δ::KanMX-FUS1-GFP
  4. Digestion of p426 Gal
Experimental steps: < Transform PCR product into FUS1-GFP strain> Consult the experiment record <2015.7.3 Experiment Record> Because last time , negative control also had grown colony so we did transformation again. At the same time , we selected colony on selection plate to do PCR check.

< Extraction of gDNA of yeast>
Consult the protocol < protocol of fast extraction of gDNA of yeast>

< Check PCR for Far1Δ::KanMX- FUS-GFP >

1.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 46℃30s
Step4→step2 for 30 cycle 72℃ 90sec
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr

2.PCR reagent
10x Dream Taq buffer 2.5μl
2.5mM dNTP0.5μl
10mM primer(F)0.5μl
10mM primer(R)0.5μl
template (First round PCR product) 8μl
Taq polymerase0.5μl
ddH2O 12.5μl
Total volume25μl
far1Δ::KanMX-FUS-GFP 260/280 260/230 ng/μl
1 1.76 0.91 111.7
2 2.05 1.32 158.2
3 2.02 1.29 120.4
4 1.98 1.08 89.9
5 2.05 0.95 66.9
6 2.07 1.45 182.6
7 1.86 0.90 95.4
8 2.08 1.45 166.4
9 2.18 0.42 22.5
10 2.07 1.30 102.4


< Digestion of p426 Gal >
  Eco RI(μl) Bam HI(μl) Uncut(μl)
ddH2O 20.5 20.5 21.5
10x NEB buf. #4 2.5 2.5 2.5
DNA(200ng) 1.5 1.5 1.5
Enzyme 0.5 0.5 --
total 25 25 25

1. Incubate at 37 for 1 hr.
2. Run the sample on the 1% agarose gel at 100 V for 30 min.(10 μl sample +2 μl loading dye)

2015.7.23

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Digestion of p426 Gal and rho-CXCR1
  2. Gel extraction of p426 Gal(vector)
  3. Clean up of rho-CXCR1(insert)
Experimental steps:
< Digestion of p426 Gal and rho-CXCR1>
  121 ng/μl 131 ng/μl
p426 Gal (2 μg) 16.5μl 15.2μl
10x NEB buf. 4 5μl 5μl
Bam HI 2μl 2μl
Eco RI 2μl 2μl
ddH2O 24.5μl 25.8μl
Total 50μl 50 μl

Rho-CXCR1 8(400 ng)
10x NEB buf. 4 5μl
Bam HI 1μl
Eco RI 1μl
ddH2O 35μl
Total 50μl

< Gel extraction of p426 Gal >
Consult the protocol < protocol of gel extraction>

< Clean up of rho-CXCR1>
Consult the protocol

2015.7.24

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Ligation of the rho-CXCR1 and p426 Gal
  2. Transform the ligation product into the E.coli
Experimental steps:
< Ligation of the rho-CXCR1 and p426 Gal>
  1:3 Vector only
Vector(11.1 ng/μl) 10μl 10μl
Insert(9.1 ng/μl) 7μl 0μl
Ligase 1μl 1μl
Ligation buff. 2μl 2μl
ddH2O 0μl 7μl
total 20μl 20μl
Incubate the ligation product at R.T. for 2 hr.

< Transform the ligation product into the competent cell>
Consult the protocol < protocol of ligation and transformation of E.coli>

2015.7.27

Operator: Wan-Yun, Jin-Ting
Goal:
  1. Fast extraction of gDNA of Far1Δ::KanMX-FUS1-GFP
  2. Check PCR for far1Δ::KanMX-FUS-GFP
Experimental steps: < Fast extraction of gDNA of far1Δ::KanMX-FUS-GFP > Consult the protocol
Strains 260/280 260/230 C(ng/μl)
FAR1∆::KANMX 1.85 1.19 177.3
FAR1∆::KANMX 1.83 1.19 163.8
Positive control 1.96 1.61 235.3

< Check PCR for Far1Δ::KanMX-FUS1-GFP>
1.PCR program
Step Temperature Time
Step1 95℃ 5min
Step2 95℃ 30s
Step3 46℃30s
Step4→step2 for 30 cycle 72℃ 90sec
Step5 72℃ 5min
Step6 (hold on) 10℃ 1hr


2.PCR reagent
  FAR1∆::KANMX Positive control
10x Dream Taq buffer 2.5μl2.5μl
2.5mM dNTP0.5μl0.5μl
10mM primer(F)0.5μl0.5μl
10mM primer(R)0.5μl 0.5μl
template (First round PCR product) 0.92μl0.64μl
Taq polymerase0.5μl0.5μl
ddH2O 19.58μl19.86μl
Total volume25μl25μl

2015.7.28

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1.Fast extraction of pGAL426 rho-CXCR1
Experimental steps:
< Fast extraction of plasmid DNA >
  1. Add 50 μl of STE buffer(100 mM NaCl, 10 mM Tris buffer, pH 7.0, 1 mM EDTA) into each new Eppendorf tube.
  2. Add 50 μl of Phenol chloroform and vortex vigorously for 30 sec.
  3. Centrifuge at 13,000 g for 5 min.
  4. Remove the 10 μl of supernatant to a new Eppendorf tube and run the sample on the 1 % agarose gel.

2015.7.29

Operator: Wan-Yun, Jin-Ting
Goal:
  1. Miniprep of p426 Gal-rho-CXCR1
  2. Enzyme digestion to check p426 Gal-rho-CXCR1
Experimental steps:
< Miniprep of p426 Gal-rho-CXCR1>
Consult the protocol

< Enzyme digestion for the p426 Gal-rho-CXCR1 check >
1. Enzyme digestion
p426 Gal-rho-CXCR1 #7,9 +,#8,12,16
10x NEB buff.4 2.5μl 2.5μl
Eco RI 0.5μl 0.5μl
Bam HI 0.5μl 0.5μl
DNA 2μl 1μl
ddH2O 19.5μl 20.5μl
total 25μl 25μl


2. Loading 10 μl sample and 2 μl DNA loading dye into the 1 % agarose gel.

2015.7.30

Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
  1. Transform pSB1C3 BBa_J04450 into the competent cell
  2. Transform the plasmid (pRS405) into the competent cell
Experimental steps:
< Transform pSB1C3 BBa_J04450 into the competent cell>
Consult the protocol
Selection plate: LB+ Cam plate

< Transform the plasmids (pRS405) into the competent cell>
Selection plate: LB+ Amp plate

Toehold Switch As RNA Sensor

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