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

Revision as of 13:18, 17 September 2015

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Description

Yeast With IL-8 Receptor

Toehold Switch As RNA Senor

Prototype 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


Name	Pur.	Seq.(5’-3’)	Size (mer.)	MW (g/mol)	Tm (℃)	GC (%)	Nmole	μl for 100μM
dFAR1 F’	Desalt	ggTTTTgTTAggCgggCAAg	20	6244.1	53.8	55	21.25	212.50
dFAR1 R’	Desalt	CATTAACTgCTATTTACgACgC	22	6669.4	51.1	41	17.12	171.20

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 dNTP	0.5μl
10mM primer(F)	0.5μl
10mM primer(R)	0.5μl
template (Far1∆::KANMX strain gDNA)	3.4μl
Taq polymerase	0.5μl
ddH2O	17.1μl
Total volume	25μl

< 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


Name	Pur.	Seq.(5’-3’)	Size (mer.)	MW (g/mol)	Tm (℃)	GC (%)	Nmole	μl for 100μM
dFAR1 F’	Desalt	ggTTTTgTTAggCgggCAAg	20	6244.1	53.8	55	21.25	212.50
dFAR1 R’	Desalt	CATTAACTgCTATTTACgACgC	22	6669.4	51.1	41	17.12	171.20

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 dNTP	0.5μl
10mM primer(F)	0.5μl
10mM primer(R)	0.5μl
template (Far1∆::KANMX strain gDNA)	3.4μl
Taq polymerase	0.5μl
ddH2O	17.1μl
Total volume	25μl

< 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 (%)	Nmole	μl for 100μM
	55	21.25	212.50
	41	17.12	171.20

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 (%)	Nmole	μl for 100μM
	55	21.25	212.50
	41	17.12	171.20

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 (%)	Nmole	μl for 100μM
	55	21.25	212.50
	41	17.12	171.20

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 (%)	Nmole	μl for 100μM
	55	21.25	212.50
	41	17.12	171.20

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 (%)	Nmole	μl for 100μM
	55	21.25	212.50
	41	17.12	171.20

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 (%)	Nmole	μl for 100μM
	55	21.25	212.50
	41	17.12	171.20

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 (%)	Nmole	μl for 100μM
	55	21.25	212.50
	41	17.12	171.20

@@ Line 132: / Line 132: @@
 																	<tr><td>ddH2O </td><td>17.1μl</td></tr>
 																	<tr><td>Total volume</td><td>25μl</td></tr>
-																</table>
+																</table><br>
 																<br>
 																< Electrophoresis to check PCR product><br>
@@ Line 145: / Line 145: @@
 																<!--照片-->
-																M：Marker；#1：Far1 ∆ for annealing at 52℃<br>
+																&nbsp;&nbsp;M：Marker；#1：Far1 ∆ for annealing at 52℃<br>
-																There is no band appears in the gel electrophoresis.<br>
+																&nbsp;&nbsp;There is no band appears in the gel electrophoresis.<br>
 																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.
+																&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.
@@ Line 224: / Line 224: @@
 																	<tr><td>ddH2O </td><td>17.1μl</td></tr>
 																	<tr><td>Total volume</td><td>25μl</td></tr>
-																</table>
+																</table><br>
+																<br>
 																< Electrophoresis to check first round-PCR product (25μl ul)><br>
 																Material:<br>