Team:CGU Taiwan/Notebook
GPCR
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
1. Design of primers
2. PCR program
3.PCR reagent
< 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.
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.
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
1. Information of primers
2.PCR program
3.PCR reagent
< 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)
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 |
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)
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
1. Design of primers
| Strains | 260/280 | 260/230 | C(ng/μl) |
| Far1∆::KANMX strain | 1.62 | 0.97 | 44.7 |
| Tm (℃) | GC (%) | Nmole | μl for 100μM | |||||
| 55 | 21.25 | 212.50 | ||||||
| 41 | 17.12 | 171.20 |
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
1. Design of primers
| Strains | 260/280 | 260/230 | C(ng/μl) |
| Far1∆::KANMX strain | 1.62 | 0.97 | 44.7 |
| Tm (℃) | GC (%) | Nmole | μl for 100μM | |||||
| 55 | 21.25 | 212.50 | ||||||
| 41 | 17.12 | 171.20 |
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
1. Design of primers
| Strains | 260/280 | 260/230 | C(ng/μl) |
| Far1∆::KANMX strain | 1.62 | 0.97 | 44.7 |
| Tm (℃) | GC (%) | Nmole | μl for 100μM | |||||
| 55 | 21.25 | 212.50 | ||||||
| 41 | 17.12 | 171.20 |
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
1. Design of primers
| Strains | 260/280 | 260/230 | C(ng/μl) |
| Far1∆::KANMX strain | 1.62 | 0.97 | 44.7 |
| Tm (℃) | GC (%) | Nmole | μl for 100μM | |||||
| 55 | 21.25 | 212.50 | ||||||
| 41 | 17.12 | 171.20 |
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
1. Design of primers
| Strains | 260/280 | 260/230 | C(ng/μl) |
| Far1∆::KANMX strain | 1.62 | 0.97 | 44.7 |
| Tm (℃) | GC (%) | Nmole | μl for 100μM | |||||
| 55 | 21.25 | 212.50 | ||||||
| 41 | 17.12 | 171.20 |
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
1. Design of primers
| Strains | 260/280 | 260/230 | C(ng/μl) |
| Far1∆::KANMX strain | 1.62 | 0.97 | 44.7 |
| Tm (℃) | GC (%) | Nmole | μl for 100μM | |||||
| 55 | 21.25 | 212.50 | ||||||
| 41 | 17.12 | 171.20 |
RNA
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
1. Design of primers
| Strains | 260/280 | 260/230 | C(ng/μl) |
| Far1∆::KANMX strain | 1.62 | 0.97 | 44.7 |
| Tm (℃) | GC (%) | Nmole | μl for 100μM | |||||
| 55 | 21.25 | 212.50 | ||||||
| 41 | 17.12 | 171.20 |