Team:CGU Taiwan/Notebook
Lab Note
Yeast With IL-8 Receptor
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)
1.PCR program
2.PCR reagent
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)
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 dNTP | 1μl |
| 10mM primer(F) | 1μl |
| 10mM primer(R) | 1μl |
| template (First round PCR product) | 1μl |
| Taq polymerase | 1μl |
| ddH2O | 40μl |
| Total volume | 50μl |
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
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:
M: Marker; #1: FAR1∆ annealing at 46 ℃
Consult the protocol < protocol of yeast transformation>
Use strain name: FUS1-GFP(His)
Selection plate: YPD+G418
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.
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:
< 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.
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>
Goal:
1. Transform PCR product into FUS::GFP (His) strain
Experiment steps:
Consult the experiment record <2015.7.3 Experiment Record>
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
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 | |
| P426GAL1/1 | 130.9ng/μl | 1.91 | 2.36 |
| P426GAL1/2 | 121.3ng/μl | 1.89 | 2.26 |
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>
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>
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>
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>
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
2.PCR reagent
< Phenol chloroform and EtOH precipitation of 2nd round PCR product >
Consult the protocol
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 dNTP | 1μl |
| 10mM primer(F) | 1μl |
| 10mM primer(R) | 1μl |
| template (First round PCR product) | 1μl |
| Taq polymerase | 1μl |
| ddH2O | 40μl |
| Total volume | 50μl |
< Phenol chloroform and EtOH precipitation of 2nd round PCR product >
Consult the protocol
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
2.PCR reagent
< Digestion of p426 Gal >
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)
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 dNTP | 0.5μl |
| 10mM primer(F) | 0.5μl |
| 10mM primer(R) | 0.5μl |
| template (First round PCR product) | 8μl |
| Taq polymerase | 0.5μl |
| ddH2O | 12.5μl |
| Total volume | 25μl |
| 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)
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>
< Gel extraction of p426 Gal >
Consult the protocol < protocol of gel extraction>
< Clean up of rho-CXCR1>
Consult the protocol
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
Toehold Switch As RNA Sensor
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 |