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:
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.
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
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>
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>
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 |
< Transform the ligation product into the competent cell>
Consult the protocol < protocol of ligation and transformation of E.coli>
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
< Check PCR for Far1Δ::KanMX-FUS1-GFP>
1.PCR program
2.PCR reagent
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 | 2.5μl | 2.5μl |
| 2.5mM dNTP | 0.5μl | 0.5μl |
| 10mM primer(F) | 0.5μl | 0.5μl |
| 10mM primer(R) | 0.5μl | 0.5μl |
| template (First round PCR product) | 0.92μl | 0.64μl |
| Taq polymerase | 0.5μl | 0.5μl |
| ddH2O | 19.58μl | 19.86μl |
| Total volume | 25μl | 25μl |
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.
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.
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
2. Loading 10 μl sample and 2 μl DNA loading dye into the 1 % agarose gel.![]()
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.
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
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
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Miniprep of pRS405 and run the 1% agarose gel
2. Digestion of pRS405 with Xho I, Bam HI and Eco RI
3. Digestion of p426 Gal with Xho I
4. Inoculate the bacteria with BBa_J04450 into the LB+ chloramphanicle broth
Experiment steps:
< Miniprep of pRS405 and run the 1% agarose gel >
Consult the protocol < protocol of miniprep plamid>
< Digestion of pRS405 with Xho I, Bam HI and Eco RI >
Reaction condition: 37℃, 1hr
< Digestion of p426 Gal with Xho I >
Reaction condition: 37℃, 1hr
Goal:
1. Miniprep of pRS405 and run the 1% agarose gel
2. Digestion of pRS405 with Xho I, Bam HI and Eco RI
3. Digestion of p426 Gal with Xho I
4. Inoculate the bacteria with BBa_J04450 into the LB+ chloramphanicle broth
Experiment steps:
< Miniprep of pRS405 and run the 1% agarose gel >
Consult the protocol < protocol of miniprep plamid>
< Digestion of pRS405 with Xho I, Bam HI and Eco RI >
| uncut | Xho I | Bam HI | Eco RI | |
| pRS405 (V105○1) | 0.5 | 0.5 | 0.5 | 0.5 |
| 10x NEB buff.4 | 2.5 | 2.5 | 2.5 | 2.5 |
| Enzyme | - | 0.5 | 0.5 | 0.5 |
| ddH2O | 22.5 | 21.5 | 21.5 | 21.5 |
| total | 25 μl | 25 μl | 25 μl | 25 μl |
< Digestion of p426 Gal with Xho I >
| . | uncut | Xho I |
| p426 Gal (131 ng/μl) | 0.5 | 0.5 |
| 10x NEB buff.4 | 2.5 | 2.5 |
| Enzyme | - | 0.5 |
| ddH2O | 22.5 | 21.5 |
| total | 25 μl | 25 μl |
Operator: Wan-Yun, Jin-Ting
Goal:
1. 1st PCR of rho-CXCR1
2. Electrophoresis to check the PCR product
3. Miniprep of pSB1C3 BBa_J04450
Experiment steps:
<1st PCR of rho-CXCR1>
1. PCR program
2. PCR reagent
< Electrophoresis to check the PCR product>
< 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: It expected length is 1200bp and it worked.
< Miniprep of pSB1C3 BBa_J04450>
Consult the protocol
Goal:
1. 1st PCR of rho-CXCR1
2. Electrophoresis to check the PCR product
3. Miniprep of pSB1C3 BBa_J04450
Experiment steps:
<1st PCR of rho-CXCR1>
1. PCR program
| Step | Temp. | Time |
| Step1 | 95℃ | 5 min |
| Step2 | 95℃ | 30 sec |
| Step3 | 40,44,48℃ | 30 sec |
| Step4→Step2 for 30 cycle | 72℃ | 80sec |
| Step5 | 72℃ | 5 min |
| Step6(Hold on) | 10℃ | 1 hr |
2. PCR reagent
| 10x Dream Taq buffer | 2.5μl |
| 2.5mMdNTP | 0.5μl |
| 10μMprimer(F’) | 0.5μl |
| 10μMprimer(R’) | 0.5 μl |
| Rho-CXCR1 | 3μl |
| Taq polymerase | 0.5 μl |
| ddH2O | 17.5μl |
| Total volume | 25 μl |
< Electrophoresis to check the PCR product>
< 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: It expected length is 1200bp and it worked.
< Miniprep of pSB1C3 BBa_J04450>
Consult the protocol
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Electrophoresis to check p426Gal-rho-CXCR1 digestion product again
2. Do digestion of Lucy-rho-CXCR1 (insert) and p426Gal (vector)
3. Clean up digestion product of Lucy-rho-CXCR1
4. Gel extraction of digestion product of p426Gal
5. Ligation insert and vector
6. Transform the construct into competent cell
Experiment steps:
< Electrophoresis to check p426 Gal-rho-CXCR1 digestion product again>
Material:
DNA marker: 1kb ladder 6ul
DNA sample:2ul p426Gal-rho-CXCR1 digestion product(BamHI and XhoI) + 1ul 6x loading buffer
Condition:
0.5xTBE buffer 100V
Time:
30min Result:
Note: pGAL426 rho-CXCR1 enzyme digestion product with BamHI and XhoI
Reaction condition : 37℃ for 1hr
< Clean up digestion product of Lucy-rho-CXCR1>
Consult the protocol
< Gel extraction of digestion product of p426Gal>
Consult the protocol
< Ligation insert and vector>
Vector:insert=1:3
Reaction condition: Room temperature for 2-4hr
< Transform the construct into competent cell>
Selection plate: LB+Cam
Goal:
1. Electrophoresis to check p426Gal-rho-CXCR1 digestion product again
2. Do digestion of Lucy-rho-CXCR1 (insert) and p426Gal (vector)
3. Clean up digestion product of Lucy-rho-CXCR1
4. Gel extraction of digestion product of p426Gal
5. Ligation insert and vector
6. Transform the construct into competent cell
Experiment steps:
< Electrophoresis to check p426 Gal-rho-CXCR1 digestion product again>
Material:
DNA marker: 1kb ladder 6ul
DNA sample:2ul p426Gal-rho-CXCR1 digestion product(BamHI and XhoI) + 1ul 6x loading buffer
Condition:
0.5xTBE buffer 100V
Time:
30min Result:
Note: pGAL426 rho-CXCR1 enzyme digestion product with BamHI and XhoI
| Lucy-rho-CXCR1 (insert) | p426Gal (vector) | p426Gal (vector) | |
| 10x NEB buffer#4 | 5μl | 5μl | 5μl |
| BamHI | 1μl | 1μl | 1μl |
| XhoI | 1μl | 1μl | 1μl |
| DNA | 25μl (400ng) | 7μl (2ug) | 7μl |
| ddH2O | 18μl | 34μl | 34μl |
| Total volume | 50μl | 50μl | 50μl |
< Clean up digestion product of Lucy-rho-CXCR1>
Consult the protocol
| Conc | 260/280 | 260/230 |
| 10.0ng/ul | 1.95 | 0.94 |
< Gel extraction of digestion product of p426Gal>
Consult the protocol
| Conc. | 260/280 | 260/230 |
| 11.2ng/ul | 1.75 | 0.20 |
< Ligation insert and vector>
Vector:insert=1:3
| Test tube | Vector only | |
| Vector(11.2ng/ul) | 9μl | 9μl |
| Insert(10.0ng/ul) | 6μl | 0μl |
| Ligase | 1μl | 1μl |
| 10xLigation buffer | 2μl | 2μl |
| ddH2O | 2μl | 8μl |
| Total volume | 20μl | 20μl |
< Transform the construct into competent cell>
Selection plate: LB+Cam
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Obeserve colony growth to confirm transformation result
2. PCR Gpa1-Leu2
3. Electrophoresis to check PCR product
4. Second PCR round of Gpa1-Leu2 with first product
5. Extraction of DNA with Phenol/Chloroform
Experiment steps:
< Obeserve colony growth to confirm transformation result>
Select 12 colonies and incubate in LB broth(with Amp) overnight,37℃
< PCR Gpa1-Leu2>
1.PCR program
2.PCR reagent
< Electrophoresis to check second round-PCR product (50μl)>
Material:
DNA marker: 1kb ladder 6μl
DNA sample:2μl PCR product + 1μl 6x loading buffer
Condition:
0.5xTBE buffer 100V
Time:
30min
Result:
Conclution: Its expected length is 1502bp and it worked.
< Second PCR round of Gpa1-Leu2 with first product>
2. PCR reagent
< Extraction of DNA with Phenol/Chloroform>
Consult the protocol
Goal:
1. Obeserve colony growth to confirm transformation result
2. PCR Gpa1-Leu2
3. Electrophoresis to check PCR product
4. Second PCR round of Gpa1-Leu2 with first product
5. Extraction of DNA with Phenol/Chloroform
Experiment steps:
< Obeserve colony growth to confirm transformation result>
Select 12 colonies and incubate in LB broth(with Amp) overnight,37℃
< PCR Gpa1-Leu2>
1.PCR program
| Step | Temperature | Time |
| Step1 | 95℃ | 5min |
| Step2 | 95℃ | 30s |
| Step3 | 48℃,53℃,59℃,62℃ | 90s |
| Step4→step2 for 30 cycle | 72℃ | 5min |
| 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(pRS405,150ng) | 0.55μl |
| Taq polymerase | 0.5μl |
| ddH2O | 20μl |
| Total volume | 25μl |
< Electrophoresis to check second round-PCR product (50μl)>
Material:
DNA marker: 1kb ladder 6μl
DNA sample:2μl PCR product + 1μl 6x loading buffer
Condition:
0.5xTBE buffer 100V
Time:
30min
Result:
Conclution: Its expected length is 1502bp and it worked.
< Second PCR round of Gpa1-Leu2 with first product>
| Step | Temperature | Time |
| Step1 | 95℃ | 5min |
| Step2 | 95℃ | 30s |
| Step3 | 53℃ | 30s |
| Step4→step2 for 30 cycle | 72℃ | 5min |
| Step5 | 72℃ | 5min |
| Step6 (hold on) | 10℃ | 1hr |
| 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 |
< Extraction of DNA with Phenol/Chloroform>
Consult the protocol
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Miniprep of p426Gal-Lucy-rho-CXCR1
2. Electrophoresis to confirm whether transformed successfully or not
3. Digestion of p426Gal-Lucy-rho-CXCR1
4. Electrophoresis to confirm which colony been transformed successlly
5. Transform Gpa1 PCR product into FAR1::KANMX-FUS1-GFP strain
< Miniprep of p426Gal-Lucy-rho-CXCR1>
Consult the protocol
< Electrophoresis to confirm whether transformed successfully or not>
Material: DNA marker: 1kb ladder 6ul
DNA sample: plasmid 2ul + 3ul 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 40min
Result:
Note: This gel result shows size difference of plasmid transformed or untransformed.
< Digestion of p426Gal-Lucy-rho-CXCR1>
Condition: incubate at 37℃ for 1hr
< Electrophoresis to confirm which colony been transformed successlly>
Material: DNA marker: 1kb ladder 6μl
DNA sample: plasmid 5μl + 3μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:
Note: We use BamHI and XhoI to check whether the construct was built successfully.
< Transform Gpa1 PCR product into FAR1::KANMX-FUS1-GFP strain>
Consult the protocol
Yeast strain: FAR1::KANMX-FUS1-GFP
Selection plate : YPD+G418 plate
Goal:
1. Miniprep of p426Gal-Lucy-rho-CXCR1
2. Electrophoresis to confirm whether transformed successfully or not
3. Digestion of p426Gal-Lucy-rho-CXCR1
4. Electrophoresis to confirm which colony been transformed successlly
5. Transform Gpa1 PCR product into FAR1::KANMX-FUS1-GFP strain
< Miniprep of p426Gal-Lucy-rho-CXCR1>
Consult the protocol
< Electrophoresis to confirm whether transformed successfully or not>
Material: DNA marker: 1kb ladder 6ul
DNA sample: plasmid 2ul + 3ul 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 40min
Result:
Note: This gel result shows size difference of plasmid transformed or untransformed.
< Digestion of p426Gal-Lucy-rho-CXCR1>
| DNA(p426Gal-Lucy-rho-CXCR1 #2 #3 #7) | 1μl |
| BamHI | 0.5μl |
| XhoI | >0.5μl |
| 10x NEB buffer #4 | 2.5μl |
| ddH2O | 20.5μl |
| Total volume | 25μl |
Condition: incubate at 37℃ for 1hr
< Electrophoresis to confirm which colony been transformed successlly>
Material: DNA marker: 1kb ladder 6μl
DNA sample: plasmid 5μl + 3μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:
Note: We use BamHI and XhoI to check whether the construct was built successfully.
< Transform Gpa1 PCR product into FAR1::KANMX-FUS1-GFP strain>
Consult the protocol
Yeast strain: FAR1::KANMX-FUS1-GFP
Selection plate : YPD+G418 plate
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Digestion of pSB1C3 BBa_J04450
2. Electrophoresis to check digestion product
3. Digestion of pSB1C3 BBa_J04450 and rho-CXCR1
4. Gel extraction of vector and clean up insert
5. Ligation and transformation
Experiment steps:
< Digestion of pSB1C3 BBa_J04450>
Condition: at 37℃ fpr 1hr
< Electrophoresis to check digestion product>
Material: DNA marker: 1kb ladder 6μl
DNA sample: 10μl + 3μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:
Note: It was up to our expection.
< Digestion of pSB1C3 BBa_J04450 and rho-CXCR1>
Condition: at 37℃ for 1hr
< Gel extraction of vector>
Consult the protocol < protocol of gel extraction>
< Clean up the insert>
Consult the protocol < protocol of clean up after digestion>
< Ligation and transformation>
Condition: room temperature 20℃ for 2hr
< Transform the construct into competent cell>
Consult the protocol
Selection plate: LB+CAM plates
Goal:
1. Digestion of pSB1C3 BBa_J04450
2. Electrophoresis to check digestion product
3. Digestion of pSB1C3 BBa_J04450 and rho-CXCR1
4. Gel extraction of vector and clean up insert
5. Ligation and transformation
Experiment steps:
< Digestion of pSB1C3 BBa_J04450>
| Both | Only XbaI | Only SpeI | Uncut | |
| pSB1C3 BBa_J04450#3 | 1μl | 1μl | 1μl | 1μl |
| 10x NEB buffer #4 | 2.5μl | 2.5μl | 2.5μl | 0μl |
| XbaI | 0.3μl | 0.3μl | 0μl | 0μl |
| SpeI | 0.3μl | 0μl | 0.3μl | 0μl |
| ddH2O | 21μl | 21.2μl | 21.2μl | 24μl |
| Total volume | 25μl | 25μl | 25μl | 25μl |
< Electrophoresis to check digestion product>
Material: DNA marker: 1kb ladder 6μl
DNA sample: 10μl + 3μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:
Note: It was up to our expection.
< Digestion of pSB1C3 BBa_J04450 and rho-CXCR1>
| Rho-CXCR1(400ng) | BBa_J04450(2μg)x2 tubes | ||
| DNA | 8μl | 9.6μl | |
| 10x NEB buffer#4 | 5μl | 5μl | |
| XbaI | 1μl | 2μl | |
| SpeI | 1μl | 2μl | |
| ddH2O | 35μl | 31.5μl | |
| Total volume | 50μl | 50μl |
< Gel extraction of vector>
Consult the protocol < protocol of gel extraction>
| Conc. | 260/280 | 260/230 |
| 6.3ng/μl | 1.62 | 0.17 |
< Clean up the insert>
Consult the protocol < protocol of clean up after digestion>
| Conc. | 260/280 | 260/230 |
| 4.9ng/μl | 1.48 | 0.68 |
< Ligation and transformation>
| Construct | Vector only | |
| Veator | 6μl | 6μl |
| Insert | 13μl | 0μl |
| 10x ligation buffer | 2μl | 2μl |
| Ligase | 1μl | 1μl |
| ddH2O | 0.5μl | 11μl |
| Total volume | 22μl | 20μl |
< Transform the construct into competent cell>
Consult the protocol
Selection plate: LB+CAM plates
Operator: Wan-Yun, Jin-Ye
Goal:
1.Fast and dirty extraction of gDNA of Gpa1 chimera yeast
2. Do PCR to check location of Gpa1 chimera
3. Electrophoresis to check PCR product
4. Do colony PCR to check location of insert (rho-CXCR1 and Lucy-rho-CXCR1)
5. Electrophoresis to check colony PCR product
Experiment steps:
< Fast and dirty extraction of gDNA of Gpa1 chimera yeast>
Consult the protocol
1.PCR program
2.PCR reagent
< Electrophoresis to check PCR product>
Electrophoresis
Material: DNA marker: 1kb ladder 6μl
DNA sample: 2μl + 3μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:
Note: Gpa1 chimera PCR check with different annealing Tm.
< Do colony PCR to check location of insert (rho-CXCR1 and Lucy-rho-CXCR1)>
< Electrophoresis to check colony PCR product>
Material: DNA marker: 6μl 1kb ladder
DNA sample: 2μl + 4μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:
Note: The middle lane was positive control---pSB1C3 BBa_J04450 and other lanes were loaded with construct built. However , the gel result was out of our expection and we assumed that they were self-ligation.
Goal:
1.Fast and dirty extraction of gDNA of Gpa1 chimera yeast
2. Do PCR to check location of Gpa1 chimera
3. Electrophoresis to check PCR product
4. Do colony PCR to check location of insert (rho-CXCR1 and Lucy-rho-CXCR1)
5. Electrophoresis to check colony PCR product
Experiment steps:
< Fast and dirty extraction of gDNA of Gpa1 chimera yeast>
Consult the protocol
| Strain name | Conc.(ng/μl) | 260/280 | 260/230 |
| Gpa1 chimera | 277.6 | 2.00 | 1.49 |
| Far1△::KANMX-FUS1-GFP | 303.6 | 2.08 | 1.71 |
1.PCR program
| Step | Temperature | Time |
| Step1 | 95℃ | 5min |
| Step2 | 95℃ | 30s |
| Step3 | 48℃,52℃,55℃,59℃ | 30s |
| Step4→step2 for 30 cycle | 72℃ | 50sec |
| Step5 | 72℃ | 5min |
| Step6 (hold on) | 10℃ | 1hr |
2.PCR reagent
| Gpa1 chimera | FAR1△::KANMX-FUS1-GFP | |
| 10x Dream Taq buffer | 2.5μl | 2.5μl |
| 2.5mM dNTP | 0.5μl | 0.5μl |
| 10mM primer(F) | 0.5μl | 0.5μl |
| 10mM primer(R) | 0.5μl | 0.5μl |
| Template(150ng) | 0.55μl | 0.5μl |
| Taq polymerase | 0.5μl | 0.5μl |
| ddH2O | 20μl | 20μl |
| Total volume | 25μl | 25μl |
< Electrophoresis to check PCR product>
Electrophoresis
Material: DNA marker: 1kb ladder 6μl
DNA sample: 2μl + 3μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:
Note: Gpa1 chimera PCR check with different annealing Tm.
< Do colony PCR to check location of insert (rho-CXCR1 and Lucy-rho-CXCR1)>
| pSB1C3 rho-CXCR1 | pSB1C3 Lucy-rho-CXCR1 | pSB1C3 BBa_J04450#3 | |
| DNA template | Colonyx7 | Colonyx7 | 0.7 |
| 10μM VF2 primer | 0.5μl | 0.5μl0.5μl | 0.5μl |
| 10μM VR primer | 0.5μl | 0.5μl | 0.5μl |
| 2x Taq mix | 12.5μl | 12.5μl | 12.5μl |
| ddH2O | 11.5μl | 11.5μl | 10.8μl |
| Total volume | 25μl | 25μl | 25μl |
< Electrophoresis to check colony PCR product>
Material: DNA marker: 6μl 1kb ladder
DNA sample: 2μl + 4μl 6x loading buffer
Condition: 0.5xTBE buffer 100V
Time: 30min
Result:
Note: The middle lane was positive control---pSB1C3 BBa_J04450 and other lanes were loaded with construct built. However , the gel result was out of our expection and we assumed that they were self-ligation.
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1.Induction of three yeast strains with α factor
2.Observe GFP under microscope.
< Induction of three yeast strains with α factor>
Consult the protocol
< Observe GFP under microscope>
Goal:
1.Induction of three yeast strains with α factor
2.Observe GFP under microscope.
< Induction of three yeast strains with α factor>
Consult the protocol
< Observe GFP under microscope>
Operator: Jin-Ting, Jin-Ye
Goal:
1.Transform pGal426 Lucy-rho-CXCR1 into strain FAR1△::KANMX-FUS1-GFP Gpa1 chimera
Experiment step:
< Transform pGal426 Lucy-rho-CXCR1 into strain FAR1△::KANMX-FUS1-GFP Gpa1 chimera>
Consult the protocol
Selection plate: Ura-+Leu- +G418 plate
Goal:
1.Transform pGal426 Lucy-rho-CXCR1 into strain FAR1△::KANMX-FUS1-GFP Gpa1 chimera
Experiment step:
< Transform pGal426 Lucy-rho-CXCR1 into strain FAR1△::KANMX-FUS1-GFP Gpa1 chimera>
Consult the protocol
Selection plate: Ura-+Leu- +G418 plate
Operator: Jin-Ting, Jin-Ye
Goal:
1.Indution of yeast with αfactor
Experiment record:
Consult protocol
Strain:
positive control: YGR254W/YJR009C/YMR186W/YPR035W/YAL038W
Negative control: wild type
Test strain: FUS1-GFP
Goal:
1.Indution of yeast with αfactor
Experiment record:
Consult protocol
Strain:
positive control: YGR254W/YJR009C/YMR186W/YPR035W/YAL038W
Negative control: wild type
Test strain: FUS1-GFP
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1.Use galactose to induce yeast experess CXCR1
Experiment record:
Consult the protocol
Strain: FAR1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 rho-CXCR1
Goal:
1.Use galactose to induce yeast experess CXCR1
Experiment record:
Consult the protocol
Strain: FAR1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 rho-CXCR1
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Protein extraction of induced yeast
Experiment steps:
Consult the protocol
Protein sample : cell culture with different induction time(3h,4h,5h,6h,7h,8h)
Goal:
1. Protein extraction of induced yeast
Experiment steps:
Consult the protocol
Protein sample : cell culture with different induction time(3h,4h,5h,6h,7h,8h)
Operator: Jin-Ting, Jin-Ye
Goal:
1.SDS-PAGE and Western blot
Experiment steps:
Consult the protocol
Protein sample : product of protein extraction last week
Positive control : HEP G2 cell lysate （from human）
Goal:
1.SDS-PAGE and Western blot
Experiment steps:
Consult the protocol
Protein sample : product of protein extraction last week
Positive control : HEP G2 cell lysate （from human）
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1. Use galactose to induce yeast express Lucy-rho-CXCR1 and rho-CXCR1
2. Treat yeast with IL-8
Experiment steps:
< Use galactose to induce yeast express Lucy-rho-CXCR1 and rho-CXCR1>
Consult the protocol
Test strain:
①Far1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 Lucy-rho-CXCR1
②Far1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 rho-CXCR1
Medium used: YEP(Raf) and YEP(Raf/Gal)
< Treat yeast with IL-8>
1. Collect 100μl yeast with galactose induction in 3hr.
2. Add 8μl IL-8(10μlM) to cell culture.
3. Take 10μl of total to observer under microscope.
Goal:
1. Use galactose to induce yeast express Lucy-rho-CXCR1 and rho-CXCR1
2. Treat yeast with IL-8
Experiment steps:
< Use galactose to induce yeast express Lucy-rho-CXCR1 and rho-CXCR1>
Consult the protocol
Test strain:
①Far1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 Lucy-rho-CXCR1
②Far1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 rho-CXCR1
Medium used: YEP(Raf) and YEP(Raf/Gal)
< Treat yeast with IL-8>
1. Collect 100μl yeast with galactose induction in 3hr.
2. Add 8μl IL-8(10μlM) to cell culture.
3. Take 10μl of total to observer under microscope.
Operator: Wan-Yun, Jin-Ting, Jin-Ye
Goal:
1.Protein extraction of yeast with galactose induced
2.Do SDS-PAGE and Western Blot
Experiment steps:
< Protein extraction of yeast with galactose induced>
Consult the protocol < protocol of protein extraction of yeast>
Yeast strain:
①Far1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 Lucy-rho-CXCR1
②Far1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 rho-CXCR1
Sample : yeast with galactose induced by 0hr, 1hr, 2hr, 3hr, 5hr, 8hr
< Do SDS-PAGE and Western Blot>
Consult the protocol < protocol of SDS-PAGE and Western blot>
Negative control: yeast with galactose induced by 0hr
Positive control: HEPG2 cell lysate
Goal:
1.Protein extraction of yeast with galactose induced
2.Do SDS-PAGE and Western Blot
Experiment steps:
< Protein extraction of yeast with galactose induced>
Consult the protocol < protocol of protein extraction of yeast>
Yeast strain:
①Far1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 Lucy-rho-CXCR1
②Far1△::KANMX-FUS1-GFP Gpa1 chimera-pGAL426 rho-CXCR1
Sample : yeast with galactose induced by 0hr, 1hr, 2hr, 3hr, 5hr, 8hr
< Do SDS-PAGE and Western Blot>
Consult the protocol < protocol of SDS-PAGE and Western blot>
Negative control: yeast with galactose induced by 0hr
Positive control: HEPG2 cell lysate
Operator: Jin-Ting, Jin-Ye
Goal:
1.Do galactose induction of yeast
2.Treat yeast with IL-8
Experiment steps:
Consult the protocol
Strain used:
①Far1△::KANMX-FUS1-GFP Gpa1 chimera
②Far1△::KANMX-FUS1-GFP Gpa1chimera pGAL426 rho-CXCR1
③Far1△::KANMX-FUS1-GFP Gpa1 chimera pGAL 426 Lucy-rho-CXCR1
Medium used: SCM(Ura-)+Raf and SCM(Ura-)+Raf/Gal
Negative control: Far1△::KANMX-FUS1-GFP Gpa1 chimera
< Treat yeast with IL-8>
1. Collect 100μl yeast culture with galactose induced by 3hr.
2. Add 8μl IL-8 (10μM) to cell culture.
3. Take 10μl of total to observe under microscope.
Positive control: YGR254W
Negative control: Wild type yeast
Positive control 2: FAR1△::KANMX-FUS1-GFP with α factor induced
Goal:
1.Do galactose induction of yeast
2.Treat yeast with IL-8
Experiment steps:
Consult the protocol
Strain used:
①Far1△::KANMX-FUS1-GFP Gpa1 chimera
②Far1△::KANMX-FUS1-GFP Gpa1chimera pGAL426 rho-CXCR1
③Far1△::KANMX-FUS1-GFP Gpa1 chimera pGAL 426 Lucy-rho-CXCR1
Medium used: SCM(Ura-)+Raf and SCM(Ura-)+Raf/Gal
Negative control: Far1△::KANMX-FUS1-GFP Gpa1 chimera
< Treat yeast with IL-8>
1. Collect 100μl yeast culture with galactose induced by 3hr.
2. Add 8μl IL-8 (10μM) to cell culture.
3. Take 10μl of total to observe under microscope.
Positive control: YGR254W
Negative control: Wild type yeast
Positive control 2: FAR1△::KANMX-FUS1-GFP with α factor induced
Toehold Switch As RNA Sensor
D, SAT (spermidine/spermine N1-acetyltransferase EST
We use the website RNA structure to predict the secondary structure of these four toehold switches whether would have hairpin structures we want. Luckily, all of its results are match our expectation.
Sent our designed sequences to IDT.
Amplify parts DNA we need.
Receive IDT DNA.
Transform BBa_I712019.
Small scale incubation.
Mini prep BBa_I712019 (Luciferase).
Concentration=186.2 (ng/ μl)
Concentration=186.2 (ng/ μl)
Digest 4 toehold switches with EcoRI and XbaI and 4 trigger DNAs with XbaI and SpeI.
Incubate at 37˚C for 3 hr.
Incubate at 37˚C for 3 hr.
| Plasmid DNA (25 ng/ μl) | 5 μl |
| NEB #2 | 1 μl |
| EcoRI (20 U/μl) | 0.2 μl |
| XbaI (20 U/μl | 0.2 μl |
| ddH2O | 3.6 μl |
| Total volume | 10 μl |
| Plasmid DNA ( 25 ng/ μl)) | 5 μl |
| NEB #2 | 1 μl |
| EcoR I (20 U/μl) | 0.2 μl |
| SpeI (20 U/μl) | 0.2 μl |
| ddH2O | 3.6 μl |
| Total volume | 10 μl |
Do the electrophoresis of digested IDT products and gel extraction
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Restriction enzyme digestion of synthetic toehold and trigger RNA with EcoRI and SpeI. Samples were run in 1% agarose gel. Lane 1, 1kb DNA
marker；Lane 2-5, 125 ng of synthetic DNA fragment that would transcribe into toehold sensors (SAT、DUSP1、IL-1β、IL-8 ) were digested by
EcoRI and SpeI；Lane 6-9, 125 ng of DNA fragment that would transcribe into trigger RNAs (SAT、DUSP1、IL-1β、IL-8 ) were digested by
EcoRI and SpeI.
All the results are around 100 bp, are the same as our expectation.
All the results are around 100 bp, are the same as our expectation.
Ask NYMU_Taiwan team for confirmed BBa_I712019, because we mistook the wrong antibiotic and lose all the parts DNA.
Get the parts and transform parts BBa_I712019.
| Vector DNA | 2 μl |
| Competent cell (DH10β) | 100 μl |
Do small scale incubation.
MIniprep BBa_I712019.
Small scale parts plasmids BBa_I712019 (Luciferase) digestion
Incubate at 37˚C for 3 hr.
| Plasmid DNA (25 ng/ μl)) | 2.5 μl |
| NEB #2 | 1 μl |
| EcoRI (20 U/μl) | 0.2 μl |
| SpeI (20 U/μl) | 0.2 μl |
| ddH2O | 6.1 μl |
| Total volume | 10 μl |
Do the electrophoresis of BBa_I712019 (Luciferase) to check the size and condition for experiment and gel extraction to check the size.
Mini-prep trigger RNAs in pSB1AK8 backbone (total 20 tubes).
Mini-prep trigger RNAs in pSB1AK8 backbone (total 20 tubes).
Largely cut luciferase in pSB1AK8 and purify it, then we got linear luciferase.
Incubate at 37˚C for 3 hr.
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Lane 1 and 4, 3 μg of luciferase digested by EcoRI and XbaI ; Lane 2, 1K DNA marker ; Lane 3, 3 μg of luciferase not digested by EcoRI and
XbaI.In the picture, uncut plasmid is higher than the cut ones. After discussing with our advisor, we suppose that the uncut plasmid is not
functional fold, so it runs slower than the plasmid with digestion.
Ligation toehold switches with luciferase (pSB1AK8).
Ligation trigger RNAs into pSB1AK8 backbone.
| Plasmid DNA (142.5 ng/ μl) | 3 μl |
| Cut smart | 3 μl |
| XbaI (20 U/μl) | 0.6 μl |
| EcoRI (20 U/μl) | 0.6 μl |
| ddH2O | 22.8 μl |
| Total volume | 30 μl |
Ligation toehold switches with luciferase (pSB1AK8).
Ligation trigger RNAs into pSB1AK8 backbone.
Transform two kinds of construct into DH10α bacteria respectively to do mass culture.
Sent our E. coli and primer (VF2) to sequence for check our assembled result.
Have synthetic toeholds (DUSP1, SAT, IL-8) -luciferase in pSB1AK8 sequence be confirmed.
Small scale T7 promoter digestion.
Large scale T7 promoter digestion, and gel extraction.
Incubate at 37˚C for 3 hr.
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Lane 1, marker ; Lane 2, 3 μg T7 promoter digested by restriction enzyme PstI and SpeI.
| Plasmid DNA | 30 μl |
| NEB #2 | 5 μl |
| PstI (20 U/μl) | 3 μl |
| SpeI (20 U/μl) | 2.6 μl |
| ddH2O | 10 μl |
| Total volume | 50 μl |
Toehold+luciferase RNA enzyme digestion, gel extraction.
| SAT1 | 241.7 (ng/ μl) |
| DUSP | 236.2 (ng/ μl) |
| IL8 | 181.2(ng/ μl) |
Ligation trigger RNA to T7 promoter in pSB1AK8 backbone.
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The trigger RNA construct we finally complete. The structure contain T7 promoter, trigger, also Ampicillin antibiotic sequence easy for us to do
the selection.
| T7 promoter linear (119.6 ng/ μl) | 15 μl |
| Plasmid DNA | 2 μl |
| NEB #2 | 1.5 μl |
| PstI (20 U/μl) | 3 μl |
| SpeI (20 U/μl) | 3 μl |
| ddH2O | 5.5 μl |
| Total volume | 30 μl |
Synthetic toehold–luciferase (IL1β) sequences confirmation.
T7 promoter-trigger RNA (SAT) in pSB1AK8 backbone sequences confirmation success, but T7 promoter-trigger RNA (DUSP1, IL1β,IL8) in pSB 1AK8 backbone sequences confirmation failed.
T7 promoter-trigger RNA (SAT) in pSB1AK8 backbone sequences confirmation success, but T7 promoter-trigger RNA (DUSP1, IL1β,IL8) in pSB 1AK8 backbone sequences confirmation failed.
Reconstruct the T7 promoter-trigger RNA (IL8,IL1 β,DUSP) in pSB1AK8 backbone.
T7 promoter-trigger RNA(DUSP1, IL-1β,IL-8) in pSB1AK8 quick screen 6 colonies each.
1. Add100 μl lysis buffer into Eppendorf tube.
2. Use stick to take a little bit bacteria into lysis buffer.
3. Add 1:1 phenol/chloroform and shake it.
4. Centrifuge(12000g, 5mins).
5. Take 10μl to electrophoresis.
1. Add100 μl lysis buffer into Eppendorf tube.
2. Use stick to take a little bit bacteria into lysis buffer.
3. Add 1:1 phenol/chloroform and shake it.
4. Centrifuge(12000g, 5mins).
5. Take 10μl to electrophoresis.
Toehold switches digestion and ligation with pSB1C3 backbone.
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The construct of toehold switches part we done. The structure include T7 promoter, toehold switches, and reporter gene—the luciferase. What’s
more, the plasmids contain the chloramphenicol resistance gene for selection.
| Plasmid DNA (SAT1) | 45 μl |
| NEB#2 | 6 μl |
| EcoRI (20 U/μl) | 4 μl |
| PstI (20 U/μl) | 4 μl |
| ddH2O | 1 μl |
| Total volume | 60 μl |
| Plasmid DNA (DUSP1) | 45 μl |
| NEB#2 | 6 μl |
| EcoRI (20 U/μl | 4 μl |
| PstI (20 U/μl | 4 μl |
| ddH2O | 1 μl |
| Total volume | 60 μl |
| Plasmid DNA (IL8) | 45 μl |
| NEB#2 | 6 μl |
| EcoRI (20 U/μl | 4 μl |
| PstI (20 U/μl | 4 μl |
| ddH2O | 1 μl |
| Total volume | 60 μl |
| Plasmid DNA (IL1β) | 45 μl |
| NEB#2 | 6 μl |
| EcoRI (20 U/μl | 4 μl |
| PstI (20 U/μl | 4 μl |
| ddH2O | 1 μl |
| Total volume | 60 μl |
| BBa_I712074 linear | 5 μl |
| Toehold switch DNA | 4 μl |
| T4 ligase | 1 μl |
| PEG400 | 2 μl |
| Total volume | 20 μl |
Transformation
| Vector DNA (158 ng/μl) | 1 μl |
| Competent cell (DH10β) | 100 μl |
Pick single colony and do the quick screen to check if the parts are inserted.
Small scale incubation.
Do the miniprep.
DNA Concentration= 174ng/μl
DNA Concentration= 174ng/μl
Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp, CAM, Amp+CAM.
T7-trigger (IL-1 β) ligation.
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In the figure, each lane are 10 μl of PCR products, we use temperature gradient (50°C, 53°C, 56°C, 59°C, 62°C, 65°C) to test the product of
which temperature condition is suitable.
we choose the result of 53 °C for PCR. Because the product of 53 °C condition is much clearer, also much brighter then other conditions.
we choose the result of 53 °C for PCR. Because the product of 53 °C condition is much clearer, also much brighter then other conditions.
Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp+CAM .
| Toehold plasmid | 100 ng |
| Trigger plasmid | 100 ng |
| Competent cell (BL21DE3) | 100 μl |
1. Grow the overnight culture before the day of experiment.
2. Dilute 200 μl overnight culture with 10 ml LB broth and incubate for 2 hours.
3. Control the culture O.D between 0.6~0.8.
4. Spin down(6000g,3 mins) 9 ml culture in Eppendorf tube.
5. Wash the pellet with 1 ml PBS and spin down (6000 g, 3 min).
6. Add 500μl 1x luciferase passive lysis buffer and move to new mini beads beater tube with enough mini beads.
7. Put the tube into mini beads beater and shock 40 sec/time.
8. Shock 4~6 times and 2 mins on ice between every shock.
9. Spin down(12000g, 5mins)the lysate and move the supernatant into new Eppendorf tube.
10. Add 1ml 1X protein assay dye into cuvette and mix with 1μl lysate sample.
11. Put the sample into DU800 spectrometer and get the concentration data.
12. Normalize the quantity of protein(0.72 mg/ml) in each Eppendorf tube.
13. Add 50μl luciferin to each sample and put into GloMax® luminometer.
14. Get the number of luciferase activity.
2. Dilute 200 μl overnight culture with 10 ml LB broth and incubate for 2 hours.
3. Control the culture O.D between 0.6~0.8.
4. Spin down(6000g,3 mins) 9 ml culture in Eppendorf tube.
5. Wash the pellet with 1 ml PBS and spin down (6000 g, 3 min).
6. Add 500μl 1x luciferase passive lysis buffer and move to new mini beads beater tube with enough mini beads.
7. Put the tube into mini beads beater and shock 40 sec/time.
8. Shock 4~6 times and 2 mins on ice between every shock.
9. Spin down(12000g, 5mins)the lysate and move the supernatant into new Eppendorf tube.
10. Add 1ml 1X protein assay dye into cuvette and mix with 1μl lysate sample.
11. Put the sample into DU800 spectrometer and get the concentration data.
12. Normalize the quantity of protein(0.72 mg/ml) in each Eppendorf tube.
13. Add 50μl luciferin to each sample and put into GloMax® luminometer.
14. Get the number of luciferase activity.
Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp+CAM again to have another plate of PCR T7-luciferase (positive).
| Toehold plasmid | 100 ng |
| Trigger plasmid | 100 ng |
| Competent cell (BL21DE3) | 100 μl |
Luciferase assay
Cotransformation (DUSP1, IL-8, SAT) in the condition of Amp+CAM .
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Luciferase assay
Failed!!! No luciferase express.
Do the electrophoresis of negative control (T7 promoter with toehold switch).
Miniprep to prepare the toehold switch IL1β.
Luciferase assay twice.
Luciferase assay twice.
Miniprep sensors and triggers in pSB1C3 (Il8,SAT1,DUSP1) and IL1β.
Luciferase assay twice.
Luciferase assay twice.
Luciferase assay once.
Luciferase assay once
Luciferase assay twice.